https://wiki.fkkt.uni-lj.si/api.php?action=feedcontributions&feedformat=atom&user=Uros.Stupar 2026-06-28T13:08:40Z User contributions MediaWiki 1.45.3 https://wiki.fkkt.uni-lj.si/index.php?title=%C4%8Ci%C5%A1%C4%8Denje_in_karakterizacija_gama_poliglutaminske_kisline_iz_bakterije_Bacillus_licheniformis_NRC20&diff=10666 2015-06-12T00:14:30Z

<p>Uros.Stupar: /* Uvod */</p> <hr /> <div>==Uvod==<br /> Gama poliglutaminska ksilina (γ-PGA) je polimerna aminokislina, ki jo proizvajajo nekateri sevi Bacillus licheniformis. Gre za nenavaden anionski polipeptid v katerem D- in/ali L- aminokislinske enote polimerizirajo preko γ-amidne vezi med α-amino in γ-karboksilno skupino naslednjega aminokislinskega ostanka. Karboksilne stranske verige lahko kemijsko modificiramo in tako vstavimo različne bioaktivne ligande ali reguliramo funkcijo polimera. Posebnost γ-PGA je v tem, da težje pride do encimske degradacije, saj proteaze, ki se jih najpogosteje uporablja, težko prepoznajo γ-povezane glutaminske kisline. Dokazali so tudi, da je γ-PGA slab imunogen, najverjetneje zaradi svoje preproste homopolimerne strukture, podobne polisaharidom. γ-PGA ni kemično sintetiziran polimer, je varen, popolnoma biorazgradljiv ter ni toksičen za človeka. Študije so pokazale, da γ-PGA pomaga pri absorpciji kalcija v črevesju, ojača imuno-stimulativno aktivnost in izboljša antitumorsko aktivnost. Poznamo različne možnosti uporabe γ-PGA: hidrogeli z veliko sposobnostjo absorpcije vode, krioprotektanti, aditiv v hrani, faktor, ki preprečuje osteoporozo, dostava zdravil, material za imobilizacijo encimov, čiščenje odpadnih vod,... Raziskave so pokazale, da prosta karboskilna skupina v vsaki enoti predstavlja možnost za vezavo nekega zdravila. γ-PGA zato spada med kandidate za selektivno dostavo kemoterapevtskih agensov.<br /> <br /> ==Namen raziskave==<br /> Namen raziskave je izolacija in karakterizacija nove bakterije, ki producira γ-PGA, optimizacija pogojev za visoko produkcijo γ-PGA in karakterizacija očiščenega polimera.<br /> <br /> ==Potek dela in rezultati==<br /> Izbrali so 25 bakterijskih izolatov, ki so jih predhodno dali v vodno kopel na 80°C za 10 minut. S tem so odstranili vegetativne celice in pa bakterije, ki ne tvorijo spor. Ugotovili so, da 7 od 25 izbranih sevov izloča polimer γ-PGA. Najbolj uspešen pri produkciji γ-PGA je bil izolat NRC20, ki so ga izolirali iz tal v rudniku. Na plošči je zrasla zelo sluzasta kolonija, v tekočem mediju pa so izmerili zelo visoko viskoznost. Na podlagi očiščenega γ-PGA so izdelali standardno krivuljo med koncentracijo γ-PGA in relativno viskoznostjo. Zaradi dobrih rezultatov so se odločili izolat NRC20 podrobneje preučiti. Na podlagi analize 16s RNA so ugotovili, da sev spada v rod Bacillus licheniformis.<br /> Naslednji korak je bil optimizacija pogojev za produkcijo γ-PGA. Ugotovili so, da Bacillus licheniformis NRC20 za rast in produkcijo γ-PGA ne potrebuje L-glutaminske kisline, vendar pa se je v njeni prisotnosti produkcija γ-PGA močno povečala. Med seboj so primerjali pet različnih medijev in ugotovili, da je viskoznost najvišja ob uporabi bazalnega medija (vsebuje vodo, različne soli potrebne za rast bakterij, vir ogljika in vir dušika), zato so ta medij tudi uporabili kot izhodišče za nadaljno optimizacijo. Nato so spremljali rast in produkcijo γ-PGA glede na vir ogljika in vir dušika, ki so ga uporabili. Izkazalo se je, da je tako rast, kot produkcija γ-PGA najboljša v mediju, ki ima kot edini vir ogljika glukozo, najboljši vir dušika pa je L-glutaminska kislina. Potrebno je bilo še optimizirati temperaturo, pH in čas inkubacije. Rast je bila najboljša pri 30°C, pH 7 in inkubacijskem času 7 dni. Vendar pa produkcija γ-PGA ni bila najvišja pri optimalnih pogojih za rast, temveč pri 35°C, pH 7-7,5 in inkubacijskem času 4-5 dni.<br /> Sledilo je čiščenje in karakterizacija γ-PGA. Bacillus licheniformis NRC20 izloča γ-PGA izven celic, tako da je čiščenje enostaven proces, ki poteka v treh stopnjah. Najprej so s centrifugiranjem odstranili bakterijske celice, nato so z etanolom oborili polimer ter na koncu še dializo odstranili nečistoče z nizko molekulsko maso. Za karakterizacijo aminokislin v γ-PGA so uporabili tankoplastno kromatografijo. Rezultat sta bili dve lisi, ki ustrezata L-glutaminski kislini in D-glutaminski kislini, in sicer v razmerju 65:35. Da bi se prepričali ali gre res za γ-PGA so polimer analizirali še z LC-ESI MS. S to metodo so dokazali prisotnost ponavljajoče se strukture, sestavljene iz enakih enot z molekulsko maso 148,1 Da, kar ustreza velikosti glutaminske kisline. Polimeri so bili različnih dolžin, vendar so bili vsi sestavljeni izključno iz glutaminske kisline.<br /> <br /> ==Zaključek==<br /> Zaključimo lahko, da je sev Bacillus licheniformis NRC20, izoliran iz tal v rudniku, obetajoč sev za visoko učinkovito produkcijo γ-PGA, ki ima zelo širok spekter uporabe. Uporablja se za dostavo zdravil, čiščenje odpadnih vod, kot krioprotektant ter za proizvodnjo nanodelcev, ki se uporabljajo v tkivnem inženirstvu ter za uspešno dostavo kemoterapevtskih agensov pri zdravljenju raka.<br /> <br /> ==Viri==<br /> Tork, Sanaa E., Magda M. Aly, Saleha Y. Alakilli, and Madeha N. Al-Seeni. 2015. “Purification and Characterization of Gamma Poly Glutamic Acid from Newly Bacillus Licheniformis NRC20.” International Journal of Biological Macromolecules 74. Elsevier B.V.: 382–91. doi:10.1016/j.ijbiomac.2014.12.017.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=%C4%8Ci%C5%A1%C4%8Denje_in_karakterizacija_gama_poliglutaminske_kisline_iz_bakterije_Bacillus_licheniformis_NRC20&diff=10665 2015-06-12T00:04:31Z

<p>Uros.Stupar: /* Zaključek */</p> <hr /> <div>==Uvod==<br /> Gama poliglutaminska ksilina (γ-PGA) je polimerna aminokislina, ki jo proizvajajo nekateri sevi Bacillus licheniformis. Gre za nenavaden anionski polipeptid v katerem D- in/ali L- aminokislinske enote polimerizirajo preko γ-amidne vezi med α-amino in γ-karboksilno skupino naslednjega aminokislinskega ostanka. Karboksilne stranske verige lahko kemijsko modificiramo in tako vstavimo različne bioaktivne ligande ali reguliramo funkcijo polimera. Posebnost γ-PGA je v tem, da težje pride do encimske degradacije, saj proteaze, ki se jih najpogosteje uporablja, težko prepoznajo γ-povezane glutaminske kisline. Dokazali so tudi, da je γ-PGA slab imunogen, najverjetneje zaradi svoje preproste homopolimerne strukture , podobne polisaharidom. γ-PGA ni kemično sintetiziran polimer, je varen, popolnoma biorazgradljiv ter ni toksičen za človeka. Študije so pokazale, da γ-PGA pomaga pri absorpciji kalcija v črevesju, ojača imuno-stimulativno aktivnost in izboljša antitumorsko aktivnost. Poznamo različne možnosti uporabe γ-PGA: hidrogeli z veliko sposobnostjo absorpcije vode, krioprotektanti, aditiv v hrani, faktor, ki preprečuje osteoporozo, dostava zdravil, material za imobilizacijo encimov, čiščenje odpadnih vod,... Raziskave so pokazale, da prosta karboskilna skupina v vsaki enoti predstavlja možnost za vezavo nekega zdravila. γ-PGA zato spada med kandidate za selektivno dostavo kemoterapevtskih agensov.<br /> ==Namen raziskave==<br /> Namen raziskave je izolacija in karakterizacija nove bakterije, ki producira γ-PGA, optimizacija pogojev za visoko produkcijo γ-PGA in karakterizacija očiščenega polimera.<br /> <br /> ==Potek dela in rezultati==<br /> Izbrali so 25 bakterijskih izolatov, ki so jih predhodno dali v vodno kopel na 80°C za 10 minut. S tem so odstranili vegetativne celice in pa bakterije, ki ne tvorijo spor. Ugotovili so, da 7 od 25 izbranih sevov izloča polimer γ-PGA. Najbolj uspešen pri produkciji γ-PGA je bil izolat NRC20, ki so ga izolirali iz tal v rudniku. Na plošči je zrasla zelo sluzasta kolonija, v tekočem mediju pa so izmerili zelo visoko viskoznost. Na podlagi očiščenega γ-PGA so izdelali standardno krivuljo med koncentracijo γ-PGA in relativno viskoznostjo. Zaradi dobrih rezultatov so se odločili izolat NRC20 podrobneje preučiti. Na podlagi analize 16s RNA so ugotovili, da sev spada v rod Bacillus licheniformis.<br /> Naslednji korak je bil optimizacija pogojev za produkcijo γ-PGA. Ugotovili so, da Bacillus licheniformis NRC20 za rast in produkcijo γ-PGA ne potrebuje L-glutaminske kisline, vendar pa se je v njeni prisotnosti produkcija γ-PGA močno povečala. Med seboj so primerjali pet različnih medijev in ugotovili, da je viskoznost najvišja ob uporabi bazalnega medija (vsebuje vodo, različne soli potrebne za rast bakterij, vir ogljika in vir dušika), zato so ta medij tudi uporabili kot izhodišče za nadaljno optimizacijo. Nato so spremljali rast in produkcijo γ-PGA glede na vir ogljika in vir dušika, ki so ga uporabili. Izkazalo se je, da je tako rast, kot produkcija γ-PGA najboljša v mediju, ki ima kot edini vir ogljika glukozo, najboljši vir dušika pa je L-glutaminska kislina. Potrebno je bilo še optimizirati temperaturo, pH in čas inkubacije. Rast je bila najboljša pri 30°C, pH 7 in inkubacijskem času 7 dni. Vendar pa produkcija γ-PGA ni bila najvišja pri optimalnih pogojih za rast, temveč pri 35°C, pH 7-7,5 in inkubacijskem času 4-5 dni.<br /> Sledilo je čiščenje in karakterizacija γ-PGA. Bacillus licheniformis NRC20 izloča γ-PGA izven celic, tako da je čiščenje enostaven proces, ki poteka v treh stopnjah. Najprej so s centrifugiranjem odstranili bakterijske celice, nato so z etanolom oborili polimer ter na koncu še dializo odstranili nečistoče z nizko molekulsko maso. Za karakterizacijo aminokislin v γ-PGA so uporabili tankoplastno kromatografijo. Rezultat sta bili dve lisi, ki ustrezata L-glutaminski kislini in D-glutaminski kislini, in sicer v razmerju 65:35. Da bi se prepričali ali gre res za γ-PGA so polimer analizirali še z LC-ESI MS. S to metodo so dokazali prisotnost ponavljajoče se strukture, sestavljene iz enakih enot z molekulsko maso 148,1 Da, kar ustreza velikosti glutaminske kisline. Polimeri so bili različnih dolžin, vendar so bili vsi sestavljeni izključno iz glutaminske kisline.<br /> <br /> ==Zaključek==<br /> Zaključimo lahko, da je sev Bacillus licheniformis NRC20, izoliran iz tal v rudniku, obetajoč sev za visoko učinkovito produkcijo γ-PGA, ki ima zelo širok spekter uporabe. Uporablja se za dostavo zdravil, čiščenje odpadnih vod, kot krioprotektant ter za proizvodnjo nanodelcev, ki se uporabljajo v tkivnem inženirstvu ter za uspešno dostavo kemoterapevtskih agensov pri zdravljenju raka.<br /> <br /> ==Viri==<br /> Tork, Sanaa E., Magda M. Aly, Saleha Y. Alakilli, and Madeha N. Al-Seeni. 2015. “Purification and Characterization of Gamma Poly Glutamic Acid from Newly Bacillus Licheniformis NRC20.” International Journal of Biological Macromolecules 74. Elsevier B.V.: 382–91. doi:10.1016/j.ijbiomac.2014.12.017.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=%C4%8Ci%C5%A1%C4%8Denje_in_karakterizacija_gama_poliglutaminske_kisline_iz_bakterije_Bacillus_licheniformis_NRC20&diff=10664 2015-06-12T00:04:03Z

<p>Uros.Stupar: /* Zaključek */</p> <hr /> <div>==Uvod==<br /> Gama poliglutaminska ksilina (γ-PGA) je polimerna aminokislina, ki jo proizvajajo nekateri sevi Bacillus licheniformis. Gre za nenavaden anionski polipeptid v katerem D- in/ali L- aminokislinske enote polimerizirajo preko γ-amidne vezi med α-amino in γ-karboksilno skupino naslednjega aminokislinskega ostanka. Karboksilne stranske verige lahko kemijsko modificiramo in tako vstavimo različne bioaktivne ligande ali reguliramo funkcijo polimera. Posebnost γ-PGA je v tem, da težje pride do encimske degradacije, saj proteaze, ki se jih najpogosteje uporablja, težko prepoznajo γ-povezane glutaminske kisline. Dokazali so tudi, da je γ-PGA slab imunogen, najverjetneje zaradi svoje preproste homopolimerne strukture , podobne polisaharidom. γ-PGA ni kemično sintetiziran polimer, je varen, popolnoma biorazgradljiv ter ni toksičen za človeka. Študije so pokazale, da γ-PGA pomaga pri absorpciji kalcija v črevesju, ojača imuno-stimulativno aktivnost in izboljša antitumorsko aktivnost. Poznamo različne možnosti uporabe γ-PGA: hidrogeli z veliko sposobnostjo absorpcije vode, krioprotektanti, aditiv v hrani, faktor, ki preprečuje osteoporozo, dostava zdravil, material za imobilizacijo encimov, čiščenje odpadnih vod,... Raziskave so pokazale, da prosta karboskilna skupina v vsaki enoti predstavlja možnost za vezavo nekega zdravila. γ-PGA zato spada med kandidate za selektivno dostavo kemoterapevtskih agensov.<br /> ==Namen raziskave==<br /> Namen raziskave je izolacija in karakterizacija nove bakterije, ki producira γ-PGA, optimizacija pogojev za visoko produkcijo γ-PGA in karakterizacija očiščenega polimera.<br /> <br /> ==Potek dela in rezultati==<br /> Izbrali so 25 bakterijskih izolatov, ki so jih predhodno dali v vodno kopel na 80°C za 10 minut. S tem so odstranili vegetativne celice in pa bakterije, ki ne tvorijo spor. Ugotovili so, da 7 od 25 izbranih sevov izloča polimer γ-PGA. Najbolj uspešen pri produkciji γ-PGA je bil izolat NRC20, ki so ga izolirali iz tal v rudniku. Na plošči je zrasla zelo sluzasta kolonija, v tekočem mediju pa so izmerili zelo visoko viskoznost. Na podlagi očiščenega γ-PGA so izdelali standardno krivuljo med koncentracijo γ-PGA in relativno viskoznostjo. Zaradi dobrih rezultatov so se odločili izolat NRC20 podrobneje preučiti. Na podlagi analize 16s RNA so ugotovili, da sev spada v rod Bacillus licheniformis.<br /> Naslednji korak je bil optimizacija pogojev za produkcijo γ-PGA. Ugotovili so, da Bacillus licheniformis NRC20 za rast in produkcijo γ-PGA ne potrebuje L-glutaminske kisline, vendar pa se je v njeni prisotnosti produkcija γ-PGA močno povečala. Med seboj so primerjali pet različnih medijev in ugotovili, da je viskoznost najvišja ob uporabi bazalnega medija (vsebuje vodo, različne soli potrebne za rast bakterij, vir ogljika in vir dušika), zato so ta medij tudi uporabili kot izhodišče za nadaljno optimizacijo. Nato so spremljali rast in produkcijo γ-PGA glede na vir ogljika in vir dušika, ki so ga uporabili. Izkazalo se je, da je tako rast, kot produkcija γ-PGA najboljša v mediju, ki ima kot edini vir ogljika glukozo, najboljši vir dušika pa je L-glutaminska kislina. Potrebno je bilo še optimizirati temperaturo, pH in čas inkubacije. Rast je bila najboljša pri 30°C, pH 7 in inkubacijskem času 7 dni. Vendar pa produkcija γ-PGA ni bila najvišja pri optimalnih pogojih za rast, temveč pri 35°C, pH 7-7,5 in inkubacijskem času 4-5 dni.<br /> Sledilo je čiščenje in karakterizacija γ-PGA. Bacillus licheniformis NRC20 izloča γ-PGA izven celic, tako da je čiščenje enostaven proces, ki poteka v treh stopnjah. Najprej so s centrifugiranjem odstranili bakterijske celice, nato so z etanolom oborili polimer ter na koncu še dializo odstranili nečistoče z nizko molekulsko maso. Za karakterizacijo aminokislin v γ-PGA so uporabili tankoplastno kromatografijo. Rezultat sta bili dve lisi, ki ustrezata L-glutaminski kislini in D-glutaminski kislini, in sicer v razmerju 65:35. Da bi se prepričali ali gre res za γ-PGA so polimer analizirali še z LC-ESI MS. S to metodo so dokazali prisotnost ponavljajoče se strukture, sestavljene iz enakih enot z molekulsko maso 148,1 Da, kar ustreza velikosti glutaminske kisline. Polimeri so bili različnih dolžin, vendar so bili vsi sestavljeni izključno iz glutaminske kisline.<br /> <br /> ==Zaključek==<br /> Zaključimo lahko, da je sev Bacillus licheniformis NRC20, izoliran iz tal v rudniku, obetajoč sev za visoko učinkovito produkcijo γ-PGA, ki ima zelo širok spekter uporabe. Uporablja se za dostavo zdravil, čiščenje odpadnih vod, kot krioprotektant ter za proizvodnjo nanodelcev, ki se uporabljajo v tkivnem inženiringu ter za uspešno dostavo kemoterapevtskih agensov pri zdravljenju raka.<br /> <br /> ==Viri==<br /> Tork, Sanaa E., Magda M. Aly, Saleha Y. Alakilli, and Madeha N. Al-Seeni. 2015. “Purification and Characterization of Gamma Poly Glutamic Acid from Newly Bacillus Licheniformis NRC20.” International Journal of Biological Macromolecules 74. Elsevier B.V.: 382–91. doi:10.1016/j.ijbiomac.2014.12.017.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=%C4%8Ci%C5%A1%C4%8Denje_in_karakterizacija_gama_poliglutaminske_kisline_iz_bakterije_Bacillus_licheniformis_NRC20&diff=10663 2015-06-12T00:02:19Z

<p>Uros.Stupar: </p> <hr /> <div>==Uvod==<br /> Gama poliglutaminska ksilina (γ-PGA) je polimerna aminokislina, ki jo proizvajajo nekateri sevi Bacillus licheniformis. Gre za nenavaden anionski polipeptid v katerem D- in/ali L- aminokislinske enote polimerizirajo preko γ-amidne vezi med α-amino in γ-karboksilno skupino naslednjega aminokislinskega ostanka. Karboksilne stranske verige lahko kemijsko modificiramo in tako vstavimo različne bioaktivne ligande ali reguliramo funkcijo polimera. Posebnost γ-PGA je v tem, da težje pride do encimske degradacije, saj proteaze, ki se jih najpogosteje uporablja, težko prepoznajo γ-povezane glutaminske kisline. Dokazali so tudi, da je γ-PGA slab imunogen, najverjetneje zaradi svoje preproste homopolimerne strukture , podobne polisaharidom. γ-PGA ni kemično sintetiziran polimer, je varen, popolnoma biorazgradljiv ter ni toksičen za človeka. Študije so pokazale, da γ-PGA pomaga pri absorpciji kalcija v črevesju, ojača imuno-stimulativno aktivnost in izboljša antitumorsko aktivnost. Poznamo različne možnosti uporabe γ-PGA: hidrogeli z veliko sposobnostjo absorpcije vode, krioprotektanti, aditiv v hrani, faktor, ki preprečuje osteoporozo, dostava zdravil, material za imobilizacijo encimov, čiščenje odpadnih vod,... Raziskave so pokazale, da prosta karboskilna skupina v vsaki enoti predstavlja možnost za vezavo nekega zdravila. γ-PGA zato spada med kandidate za selektivno dostavo kemoterapevtskih agensov.<br /> ==Namen raziskave==<br /> Namen raziskave je izolacija in karakterizacija nove bakterije, ki producira γ-PGA, optimizacija pogojev za visoko produkcijo γ-PGA in karakterizacija očiščenega polimera.<br /> <br /> ==Potek dela in rezultati==<br /> Izbrali so 25 bakterijskih izolatov, ki so jih predhodno dali v vodno kopel na 80°C za 10 minut. S tem so odstranili vegetativne celice in pa bakterije, ki ne tvorijo spor. Ugotovili so, da 7 od 25 izbranih sevov izloča polimer γ-PGA. Najbolj uspešen pri produkciji γ-PGA je bil izolat NRC20, ki so ga izolirali iz tal v rudniku. Na plošči je zrasla zelo sluzasta kolonija, v tekočem mediju pa so izmerili zelo visoko viskoznost. Na podlagi očiščenega γ-PGA so izdelali standardno krivuljo med koncentracijo γ-PGA in relativno viskoznostjo. Zaradi dobrih rezultatov so se odločili izolat NRC20 podrobneje preučiti. Na podlagi analize 16s RNA so ugotovili, da sev spada v rod Bacillus licheniformis.<br /> Naslednji korak je bil optimizacija pogojev za produkcijo γ-PGA. Ugotovili so, da Bacillus licheniformis NRC20 za rast in produkcijo γ-PGA ne potrebuje L-glutaminske kisline, vendar pa se je v njeni prisotnosti produkcija γ-PGA močno povečala. Med seboj so primerjali pet različnih medijev in ugotovili, da je viskoznost najvišja ob uporabi bazalnega medija (vsebuje vodo, različne soli potrebne za rast bakterij, vir ogljika in vir dušika), zato so ta medij tudi uporabili kot izhodišče za nadaljno optimizacijo. Nato so spremljali rast in produkcijo γ-PGA glede na vir ogljika in vir dušika, ki so ga uporabili. Izkazalo se je, da je tako rast, kot produkcija γ-PGA najboljša v mediju, ki ima kot edini vir ogljika glukozo, najboljši vir dušika pa je L-glutaminska kislina. Potrebno je bilo še optimizirati temperaturo, pH in čas inkubacije. Rast je bila najboljša pri 30°C, pH 7 in inkubacijskem času 7 dni. Vendar pa produkcija γ-PGA ni bila najvišja pri optimalnih pogojih za rast, temveč pri 35°C, pH 7-7,5 in inkubacijskem času 4-5 dni.<br /> Sledilo je čiščenje in karakterizacija γ-PGA. Bacillus licheniformis NRC20 izloča γ-PGA izven celic, tako da je čiščenje enostaven proces, ki poteka v treh stopnjah. Najprej so s centrifugiranjem odstranili bakterijske celice, nato so z etanolom oborili polimer ter na koncu še dializo odstranili nečistoče z nizko molekulsko maso. Za karakterizacijo aminokislin v γ-PGA so uporabili tankoplastno kromatografijo. Rezultat sta bili dve lisi, ki ustrezata L-glutaminski kislini in D-glutaminski kislini, in sicer v razmerju 65:35. Da bi se prepričali ali gre res za γ-PGA so polimer analizirali še z LC-ESI MS. S to metodo so dokazali prisotnost ponavljajoče se strukture, sestavljene iz enakih enot z molekulsko maso 148,1 Da, kar ustreza velikosti glutaminske kisline. Polimeri so bili različnih dolžin, vendar so bili vsi sestavljeni izključno iz glutaminske kisline.<br /> <br /> ==Zaključek==<br /> Zaključimo lahko, da je sev Bacillus licheniformis NRC20, izoliran iz tal v rudniku, obetajoč sev za visoko učinkovito produkcijo γ-PGA, ki ima zelo širok spekter uporabe. Uporablje se za dostavo zdravil, čiščenje odpadnih vod, kot krioprotektant ter za proizvodnjo nanodelcev, ki se uporabljajo v tkivnem inženiringu ter za uspešno dostavo kemoterapevtskih agensov pri zdravljenju raka.<br /> <br /> ==Viri==<br /> Tork, Sanaa E., Magda M. Aly, Saleha Y. Alakilli, and Madeha N. Al-Seeni. 2015. “Purification and Characterization of Gamma Poly Glutamic Acid from Newly Bacillus Licheniformis NRC20.” International Journal of Biological Macromolecules 74. Elsevier B.V.: 382–91. doi:10.1016/j.ijbiomac.2014.12.017.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=%C4%8Ci%C5%A1%C4%8Denje_in_karakterizacija_gama_poliglutaminske_kisline_iz_bakterije_Bacillus_licheniformis_NRC20&diff=10662 2015-06-11T23:58:14Z

<p>Uros.Stupar: /* Namen raziskave */</p> <hr /> <div>==Uvod==<br /> Gama poliglutaminska ksilina (γ-PGA) je polimerna aminokislina, ki jo proizvajajo nekateri sevi Bacillus licheniformis. Gre za nenavaden anionski polipeptid v katerem D- in/ali L- aminokislinske enote polimerizirajo preko γ-amidne vezi med α-amino in γ-karboksilno skupino naslednjega aminokislinskega ostanka. Karboksilne stranske verige lahko kemijsko modificiramo in tako vstavimo različne bioaktivne ligande ali reguliramo funkcijo polimera. Posebnost γ-PGA je v tem, da težje pride do encimske degradacije, saj proteaze, ki se jih najpogosteje uporablja, težko prepoznajo γ-povezane glutaminske kisline. Dokazali so tudi, da je γ-PGA slab imunogen, najverjetneje zaradi svoje preproste homopolimerne strukture , podobne polisaharidom. γ-PGA ni kemično sintetiziran polimer, je varen, popolnoma biorazgradljiv ter ni toksičen za človeka. Študije so pokazale, da γ-PGA pomaga pri absorpciji kalcija v črevesju, ojača imuno-stimulativno aktivnost in izboljša antitumorsko aktivnost. Poznamo različne možnosti uporabe γ-PGA: hidrogeli z veliko sposobnostjo absorpcije vode, krioprotektanti, aditiv v hrani, faktor, ki preprečuje osteoporozo, dostava zdravil, material za imobilizacijo encimov, čiščenje odpadnih vod,... Raziskave so pokazale, da prosta karboskilna skupina v vsaki enoti predstavlja možnost za vezavo nekega zdravila. γ-PGA zato spada med kandidate za selektivno dostavo kemoterapevtskih agensov.<br /> ==Namen raziskave==<br /> Namen raziskave je izolacija in karakterizacija nove bakterije, ki producira γ-PGA, optimizacija pogojev za visoko produkcijo γ-PGA in karakterizacija očiščenega polimera.<br /> <br /> ==Potek dela in rezultati==<br /> Izbrali so 25 bakterijskih izolatov, ki so jih predhodno dali v vodno kopel na 80°C za 10 minut. S tem so odstranili vegetativne celice in pa bakterije, ki ne tvorijo spor. Ugotovili so, da 7 od 25 izbranih sevov izloča polimer γ-PGA. Najbolj uspešen pri produkciji γ-PGA je bil izolat NRC20, ki so ga izolirali iz tal v rudniku. Na plošči je zrasla zelo sluzasta kolonija, v tekočem mediju pa so izmerili zelo visoko viskoznost. Na podlagi očiščenega γ-PGA so izdelali standardno krivuljo med koncentracijo γ-PGA in relativno viskoznostjo. Zaradi dobrih rezultatov so se odločili izolat NRC20 podrobneje preučiti. Na podlagi analize 16s RNA so ugotovili, da sev spada v rod Bacillus licheniformis.<br /> Naslednji korak je bil optimizacija pogojev za produkcijo γ-PGA. Ugotovili so, da Bacillus licheniformis NRC20 za rast in produkcijo γ-PGA ne potrebuje L-glutaminske kisline, vendar pa se je v njeni prisotnosti produkcija γ-PGA močno povečala. Med seboj so primerjali pet različnih medijev in ugotovili, da je viskoznost najvišja ob uporabi bazalnega medija (vsebuje vodo, različne soli potrebne za rast bakterij, vir ogljika in vir dušika), zato so ta medij tudi uporabili kot izhodišče za nadaljno optimizacijo. Nato so spremljali rast in produkcijo γ-PGA glede na vir ogljika in vir dušika, ki so ga uporabili. Izkazalo se je, da je tako rast, kot produkcija γ-PGA najboljša v mediju, ki ima kot edini vir ogljika glukozo, najboljši vir dušika pa je L-glutaminska kislina. Potrebno je bilo še optimizirati temperaturo, pH in čas inkubacije. Rast je bila najboljša pri 30°C, pH 7 in inkubacijskem času 7 dni. Vendar pa produkcija γ-PGA ni bila najvišja pri optimalnih pogojih za rast, temveč pri 35°C, pH 7-7,5 in inkubacijskem času 4-5 dni.<br /> Sledilo je čiščenje in karakterizacija γ-PGA. Bacillus licheniformis NRC20 izloča γ-PGA izven celic, tako da je čiščenje enostaven proces, ki poteka v treh stopnjah. Najprej so s centrifugiranjem odstranili bakterijske celice, nato so z etanolom oborili polimer ter na koncu še dializo odstranili nečistoče z nizko molekulsko maso. Za karakterizacijo aminokislin v γ-PGA so uporabili tankoplastno kromatografijo. Rezultat sta bili dve lisi, ki ustrezata L-glutaminski kislini in D-glutaminski kislini, in sicer v razmerju 65:35. Da bi se prepričali ali gre res za γ-PGA so polimer analizirali še z LC-ESI MS. S to metodo so dokazali prisotnost ponavljajoče se strukture, sestavljene iz enakih enot z molekulsko maso 148,1 Da, kar ustreza velikosti glutaminske kisline. Polimeri so bili različnih dolžin, vendar so bili vsi sestavljeni izključno iz glutaminske kisline.<br /> <br /> ==Zaključek==<br /> Zaključimo lahko, da je sev Bacillus licheniformis NRC20, izoliran iz tal v rudniku, obetajoč sev za visoko učinkovito produkcijo γ-PGA, ki ima zelo širok spekter uporabe. Uporablje se za dostavo zdravil, čiščenje odpadnih vod, kot krioprotektant ter za proizvodnjo nanodelcev, ki se uporabljajo v tkivnem inženiringu ter za uspešno dostavo kemoterapevtskih agensov pri zdravljenju raka.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=%C4%8Ci%C5%A1%C4%8Denje_in_karakterizacija_gama_poliglutaminske_kisline_iz_bakterije_Bacillus_licheniformis_NRC20&diff=10661 2015-06-11T23:57:50Z

<p>Uros.Stupar: New page: ==Uvod== Gama poliglutaminska ksilina (γ-PGA) je polimerna aminokislina, ki jo proizvajajo nekateri sevi Bacillus licheniformis. Gre za nenavaden anionski polipeptid v katerem D- in/ali ...</p> <hr /> <div>==Uvod==<br /> Gama poliglutaminska ksilina (γ-PGA) je polimerna aminokislina, ki jo proizvajajo nekateri sevi Bacillus licheniformis. Gre za nenavaden anionski polipeptid v katerem D- in/ali L- aminokislinske enote polimerizirajo preko γ-amidne vezi med α-amino in γ-karboksilno skupino naslednjega aminokislinskega ostanka. Karboksilne stranske verige lahko kemijsko modificiramo in tako vstavimo različne bioaktivne ligande ali reguliramo funkcijo polimera. Posebnost γ-PGA je v tem, da težje pride do encimske degradacije, saj proteaze, ki se jih najpogosteje uporablja, težko prepoznajo γ-povezane glutaminske kisline. Dokazali so tudi, da je γ-PGA slab imunogen, najverjetneje zaradi svoje preproste homopolimerne strukture , podobne polisaharidom. γ-PGA ni kemično sintetiziran polimer, je varen, popolnoma biorazgradljiv ter ni toksičen za človeka. Študije so pokazale, da γ-PGA pomaga pri absorpciji kalcija v črevesju, ojača imuno-stimulativno aktivnost in izboljša antitumorsko aktivnost. Poznamo različne možnosti uporabe γ-PGA: hidrogeli z veliko sposobnostjo absorpcije vode, krioprotektanti, aditiv v hrani, faktor, ki preprečuje osteoporozo, dostava zdravil, material za imobilizacijo encimov, čiščenje odpadnih vod,... Raziskave so pokazale, da prosta karboskilna skupina v vsaki enoti predstavlja možnost za vezavo nekega zdravila. γ-PGA zato spada med kandidate za selektivno dostavo kemoterapevtskih agensov.<br /> ==Namen raziskave==<br /> Namen te raziskave je izolacija in karakterizacija nove bakterije, ki producira γ-PGA, optimizacija pogojev za visoko produkcijo γ-PGA in karakterizacija očiščenega polimera. <br /> ==Potek dela in rezultati==<br /> Izbrali so 25 bakterijskih izolatov, ki so jih predhodno dali v vodno kopel na 80°C za 10 minut. S tem so odstranili vegetativne celice in pa bakterije, ki ne tvorijo spor. Ugotovili so, da 7 od 25 izbranih sevov izloča polimer γ-PGA. Najbolj uspešen pri produkciji γ-PGA je bil izolat NRC20, ki so ga izolirali iz tal v rudniku. Na plošči je zrasla zelo sluzasta kolonija, v tekočem mediju pa so izmerili zelo visoko viskoznost. Na podlagi očiščenega γ-PGA so izdelali standardno krivuljo med koncentracijo γ-PGA in relativno viskoznostjo. Zaradi dobrih rezultatov so se odločili izolat NRC20 podrobneje preučiti. Na podlagi analize 16s RNA so ugotovili, da sev spada v rod Bacillus licheniformis.<br /> Naslednji korak je bil optimizacija pogojev za produkcijo γ-PGA. Ugotovili so, da Bacillus licheniformis NRC20 za rast in produkcijo γ-PGA ne potrebuje L-glutaminske kisline, vendar pa se je v njeni prisotnosti produkcija γ-PGA močno povečala. Med seboj so primerjali pet različnih medijev in ugotovili, da je viskoznost najvišja ob uporabi bazalnega medija (vsebuje vodo, različne soli potrebne za rast bakterij, vir ogljika in vir dušika), zato so ta medij tudi uporabili kot izhodišče za nadaljno optimizacijo. Nato so spremljali rast in produkcijo γ-PGA glede na vir ogljika in vir dušika, ki so ga uporabili. Izkazalo se je, da je tako rast, kot produkcija γ-PGA najboljša v mediju, ki ima kot edini vir ogljika glukozo, najboljši vir dušika pa je L-glutaminska kislina. Potrebno je bilo še optimizirati temperaturo, pH in čas inkubacije. Rast je bila najboljša pri 30°C, pH 7 in inkubacijskem času 7 dni. Vendar pa produkcija γ-PGA ni bila najvišja pri optimalnih pogojih za rast, temveč pri 35°C, pH 7-7,5 in inkubacijskem času 4-5 dni.<br /> Sledilo je čiščenje in karakterizacija γ-PGA. Bacillus licheniformis NRC20 izloča γ-PGA izven celic, tako da je čiščenje enostaven proces, ki poteka v treh stopnjah. Najprej so s centrifugiranjem odstranili bakterijske celice, nato so z etanolom oborili polimer ter na koncu še dializo odstranili nečistoče z nizko molekulsko maso. Za karakterizacijo aminokislin v γ-PGA so uporabili tankoplastno kromatografijo. Rezultat sta bili dve lisi, ki ustrezata L-glutaminski kislini in D-glutaminski kislini, in sicer v razmerju 65:35. Da bi se prepričali ali gre res za γ-PGA so polimer analizirali še z LC-ESI MS. S to metodo so dokazali prisotnost ponavljajoče se strukture, sestavljene iz enakih enot z molekulsko maso 148,1 Da, kar ustreza velikosti glutaminske kisline. Polimeri so bili različnih dolžin, vendar so bili vsi sestavljeni izključno iz glutaminske kisline.<br /> <br /> ==Zaključek==<br /> Zaključimo lahko, da je sev Bacillus licheniformis NRC20, izoliran iz tal v rudniku, obetajoč sev za visoko učinkovito produkcijo γ-PGA, ki ima zelo širok spekter uporabe. Uporablje se za dostavo zdravil, čiščenje odpadnih vod, kot krioprotektant ter za proizvodnjo nanodelcev, ki se uporabljajo v tkivnem inženiringu ter za uspešno dostavo kemoterapevtskih agensov pri zdravljenju raka.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=MBT_seminarji_2015&diff=10660 2015-06-11T23:52:25Z

<p>Uros.Stupar: </p> <hr /> <div>'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2014/15'''<br /> <br /> Tabela za razpored po tednih bo objavljena v spletni učilnici, vanjo pa se vpišite tudi za kratke predstavitve novic (3 min, dvakrat v semestru). Na tej strani bo samo seznam odobrenih člankov za seminar in povezave do člankov in do povzetkov, ki jih morate objaviti najkasneje tri dni pred predstavitvijo (ponedeljek oz. torek). Angleški naslov prevedite tudi v slovenščino - to bo naslov povzetka, ki ga objavite na posebni strani, tako kot so to naredili kolegi pred vami (oz. lani).<br /> <br /> Način vnosa:<br /> <br /> # The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops &amp; Food 5(1), 2014; http://dx.doi.org/10.4161/gmcr.26979) Pomen glutation peroksidaze 8 iz repnjakovca za zaščito rastline ''Arabidopsis thaliana'' in bakterije ''Escherichia coli'' pred oksidativnim stresom. Janez Novak, 15. marca 2014<br /> (slovenski naslov povežite z novo stranjo, na kateri bo povzetek)<br /> <br /> <br /> '''Naslovi odobrenih člankov po temah:'''<br /> <br /> '''Gensko spremenjene rastline'''&lt;br&gt;<br /> # Successful high-level accumulation of fish oil omega-3 long-chain polyunsaturated fatty acids in a transgenic oilseed crop (Ruiz-Lopez, N., et al; The plant journal 77, 198-208, 2014; http://www.ncbi.nlm.nih.gov/pubmed/24308505). [[Uspešna priprava gensko spremenjene oljne rastline z visoko vsebnostjo omega-3 polinenasičenih maščobnih kislin.]] Petra Malavašič, 20. marca 2015<br /> #A simplified and accurate detection of the genetically modified wheat MON71800 with one calibrator plasmid (Jae Juan, S.,et al; Food Chemistry 176, 1-6, ;http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S03088146140196572015 [[Poenostavljena in točna detekcija gensko spemenjene pšenice MON71800 z enim kalibratorskim plazmidom]]. Matej Lesar, 20. marca 2015<br /> <br /> '''Gensko spremenjene živali'''&lt;br&gt;<br /> # [[A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expresion]] (H. Chen; et all.; BMC Biotechnology 2015, 15:4, doi:10.1186/s12896-015-0121-4; http://www.biomedcentral.com/1472-6750/15/4). Edvinas Grauželis, 27. marca 2015 (in English)<br /> # Production of functional active human growth factors in insects used as living biofactories (B. Dudognon, et al; Journal of Biotechnology 184, 229–239, 2014; http://dx.doi.org/10.1016/j.jbiotec.2014.05.030). [[Proizvodnja funkcionalno aktivnih človeških rastnih faktorjev v insektih uporabljenih kot žive biotovarne]] Maxi Sagmeister, 27. marca 2015<br /> <br /> '''Okolje'''&lt;br&gt;<br /> # Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles (Yuan Gao ''et al.'', Enzyme and Microbial Technology, vol. 54, pages 38-44, 10.1.2014, http://www.sciencedirect.com/science/article/pii/S0141022913002044). [[Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliestrski tekstil]]. Mitja Crček, 3. aprila 2015<br /> # Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene (A. W. Vail ''et al.''; Transgenic Research, 29. 11. 2014; http://link.springer.com/article/10.1007/s11248-014-9851-7). [[Biorazgradnja atrazina s tremi transgenskimi travami in lucerno, ki izražajo gen za modificirano bakterijsko atrazin klorohidrolazo]]. Mirjam Kmetič, 3. aprila 2015 <br /> <br /> '''Terapevtiki'''&lt;br&gt;<br /> # Glycosylated enfuvirtide: A long-lasting glycopeptide with potent anti-HIV activity; http://pubs.acs.org/doi/full/10.1021/jm5016582 [[Glikoliziran Enfuvirtid: glikopeptid z močno proti HIV aktivnostjo s podaljšanim delovanjem]]. Sebastian Pleško, 10. aprila <br /> # Microbicidal effects of α- and θ-defensins against antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa; http://ini.sagepub.com/content/21/1/17.long. [[Mikrobicidno delovanje α in θ defenzinov na antibiotik-odporne Staphylococcus aureus in Pseudomonas aeruginosa]]. Ana Kapraljević, 10. aprila<br /> <br /> '''Encimi'''&lt;br&gt;<br /> # Immobilization and controlled release of β-galactosidase from chitosan-grafted hydrogels; http://www.sciencedirect.com/science/article/pii/S0308814615001028. [[Imobilizacija in nadzorovano sproščanje β-galaktozidaze iz hitozanskega hidrogela]]. Mojca Banič, 16. aprila 2015<br /> # Construction of efficient xylose utilizing ''Pichia pastoris'' for industrial enzyme production (Li ''et al''; Microbial Cell Factories 14:22, 1-10, 2015; http://www.microbialcellfactories.com/content/14/1/22). [[Priprava Pichie pastoris, ki učinkovito uporablja ksilozo, za industrijsko proizvodnjo encimov]]. Špela Tomaž, 17. aprila 2015<br /> # Postharvest application of a novel chitinase cloned from ''Metschnikowia fructicola'' and overexpressed in ''Pichia pastoris'' to control brown rot of peaches; http://www.sciencedirect.com/science/article/pii/S0168160515000033. [[Uporaba hitinaze, klonirane iz Metschnikowie fructicola in prekomerno izražene v Pichii pastoris za nadzor rjave gnilobe breskev po obiranju]] Špela Pohleven, 17. aprila 2015<br /> <br /> '''Protitelesa'''&lt;br&gt; <br /> # Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells; http://dx.plos.org/10.1371/journal.pone.0116878. Optimizacija signalnih peptidov težkih in lahkih verig za večjo ekspresijo terapevtskih protiteles v CHO celičnih linijah. [[Optimizacija signalnih peptidov težkih in lahkih verig za večjo ekspresijo terapevtskih protiteles v CHO celičnih linijah]] Tjaša Blatnik, 23. aprila 2015<br /> # Ethanol precipitation for purification of recombinant antibodies (A. Tscheliessnig ''et al''; Journal of Biotechnology 188, 17-28, 2014; http://www.sciencedirect.com/science/article/pii/S0168165614007810). [[Čiščenje rekombinantnih protiteles z obarjanjem z etanolom]]. Urška Rauter, 24. aprila 2015<br /> # Functional mutations in and characterization of VHH against ''Helicobacter pylori'' urease (R. Hoseinpoor ''et al''; Applied Biochemistry and Biotechnology 172, 3079-3091, 2014; http://link.springer.com/article/10.1007/s12010-014-0750-4). [[Funkcionalne mutacije in karakterizacija VHH proti ureazi Helicobacter pylori]]. Marko Radojković, 7. maja 2015<br /> <br /> '''Cepiva'''&lt;br&gt;<br /> # Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions; http://www.sciencedirect.com/science/article/pii/S0378517315001507. [[Razvoj pegiliranih lipopleksov proti E6 za aplikacijo na sluznico pri predrakavih spremembah materničnega vratu]]. Tanja Korpar, 7. maja 2015<br /> # A novel “priming-boosting” strategy for immune interventions in cervical cancer (S. Liao et al.; Molecular Immunology 64, 295-305, 2015, http://www.sciencedirect.com/science/article/pii/S0161589014003460. [[Nova &quot;priming-boosting&quot; strategija za imunsko posredovanje pri raku materničnega vratu]]. Anita Kustec, 8. maja 2015<br /> # Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors (H.C. Wang et al.; Immunology letters 163, 206-213, 2015, http://www.ncbi.nlm.nih.gov/pubmed/25102364 ) [[Izboljšava cepiv proti antraksu z uporabo iz virusnih replikonov izvedenih vektorjev, ki omogočajo izražanje zaščitnega antigena.]] Daša Pavc, 8. maja 2015<br /> <br /> '''Male molekule in polimeri'''&lt;br&gt;<br /> # Methanol-induced chain termination in poly(3-hydroxybutyrate) biopolymers: Molecular weight control; http://www.sciencedirect.com/science/article/pii/S0141813014008307. [[Z metanolom inducirana terminacija polimerizacije poli(3-hidroksibutiratnih) polimerov: Vpliv na molekulsko maso]]. Gašper Lavrenčič, 14. maja 2015<br /> # Purification and characterization of gamma poly glutamic acid from newly Bacillus licheniformis NRC20; http://www.sciencedirect.com/science/article/pii/S0141813014008216. [[Čiščenje in karakterizacija gama poliglutaminske kisline iz bakterije Bacillus licheniformis NRC20]]. Uroš Stupar, 14. maja 2015<br /> # Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases (Citorik RJ. ''et al''; Nature Biotechnology 32, 1141-1145, 2014; http://www.nature.com/nbt/journal/v32/n11/full/nbt.3011.html). [[Sekven%C4%8Dno specifi%C4%8Dna protimikrobna sredstva]] Iza Ogris, 15. maja 2015<br /> # Chromosomal integration of hyaluronic acid synthesis (''has'') genes enhances the molecular weight of hyaluronan produced in ''Lactococcus lactis'' (R. V. Hmar et al; Biotechnol. J. 9 (12), 2014; http://dx.doi.org/10.1002/biot.201400215) [[Integracija genov za sintezo hialuronske kisline v kromosom bakterije Lactococcus lactis izboljša sintezo visokomolekularne hialuronske kisline]] Maja Grdadolnik, 15. maja 2015<br /> <br /> '''Pretvorba biomase'''&lt;br&gt;<br /> # Effect of pretreatment methods on the synergism of cellulase and xylanase during the hydrolysis of bagasse (L. Jia ''et al''; Bioresource Technology 185, 2015; http://www.sciencedirect.com/science/article/pii/S0960852415002114) [[Vpliv metod predobdelave na sinergizem celulaze in ksilanaze pri hidrolizi bagase]]. Eva Lucija Kozak, 21. maja 2015<br /> # Third generation biohydrogen production by Clostridium butyricum and adapted mixed cultures from Scenedesmus obliquus microalga biomass; http://www.sciencedirect.com/science/article/pii/S0016236115002550?np=y [[Tretja generacija proizvodnje biovodika s pomočjo, z mikroalgami Scenedesmus obliquus hranjenimi bakterijami Clostridium butyricum in mešanico prilagojenih mikroorganizmov]] Nives Naraglav, 22. maja 2015<br /> # Bio-catalytic action of twin-screw extruder enzymatic hydrolysis on the deconstruction of annual plant material: Case of sweet corn co-products; http://www.sciencedirect.com/science/article/pii/S0926669015000436 [[Biokatalitični učinek encimske hidrolize dvovijačnega ekstruderja na destrukturiranje rastlinskega materiala]]. Griša Prinčič, 22. maja 2015<br /> <br /> '''Metabolično inženirstvo'''&lt;br&gt;<br /> # Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica (K. Qiao ''et al.''; Metabolic Engineering 29, 2014; http://www.sciencedirect.com/science/article/pii/S1096717615000166) [[Povečanje proizvodnje lipidov v kvasovki Yarrowia lipolytica]]. Andreja Bratovš, 28. maja 2015<br /> # Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals (Weerawat Runguphana, Jay D. Keasling; Metabolic Engineering, vol 21, January 2014, Pages 103–113; http://www.sciencedirect.com/science/article/pii/S1096717613000670). [[Metabolno inženirstvo kvasovke Saccharomyces cerevisiae za proizvodnjo biogoriva in kemikalij iz maščobnih kislin]]. Dominik Kert, 29. maja 2015<br /> # Metabolic engineering of Klebsiella pneumoniae for the production of cis,cis-muconic acid (Jung,H.-M. Jung,M.-Y. Oh, M.-K.;Applied Microbiology and Biotechnology, Published online: 14 February 2015; http://link.springer.com/article/10.1007/s00253-015-6442-3). [[Metabolno inženirstvo Klebsiella pneumoniae za produkcijo cis,cis-mukonične kisline]]. Jure Zabret, 29. maja 2015<br /> <br /> '''Biološki viri energije'''&lt;br&gt;<br /> # Anodic and cathodic microbial communities in single chamber microbial fuel cells; http://www.sciencedirect.com/science/article/pii/S1871678414021694. [[Anodna in katodna mikrobna združba v eno-celični mikrobni gorivni celici]] Tamara Marić, 4. junija 2015<br /> # Combination of dry dark fermentation and mechanical pretreatment for lignocellulosic deconstruction: An innovative strategy for biofuels and volatile fatty acids recovery; http://www.sciencedirect.com/science/article/pii/S0306261915002196. [[Kombinacija temne fermentacije v trdnem stanju in mehanske obdelave za razgradnjo lignoceluloze: Inovativen pristop za proizvodnjo biogoriv in hlapnih organskih kislin.]] Jernej Pušnik, 4. junija 2015<br /> # Potential use of feedlot cattle manure for bioethanol production; http://www.sciencedirect.com/science/article/pii/S0960852415001960. [[Uporaba govejega gnoja v proizvodnji bioetanola.]] Nastja Pirman, 5. junija 2015<br /> # Cellulolytic enzymes produced by a newly isolated soil fungus Penicillium sp. TG2 with potential for use in cellulosic ethanol production; http://www.sciencedirect.com/science/article/pii/S0960148114007022. [[Celulolitični encimi talne glive Penicillium sp. TG2 in njihov potencial pri proizvodnji etanola.]] Jana Verbančič, 5. junija 2015<br /> <br /> '''Novi pristopi v molekularni biotehnologiji'''&lt;br&gt;<br /> # Exploring the potential of algae/bacteria interactions; http://www.sciencedirect.com/science/article/pii/S0958166915000269. [[Potenciali interakcij med algami in bakterijami.]] Matja Zalar, 11. junija<br /> # How close we are to achieving commercially viable large-scale photobiological hydrogen production by cyanobacteria: A review of the biological aspects; http://www.mdpi.com/2075-1729/5/1/997/htm. [[Kako blizu smo dosegu komercialno dostopne masovne fotobiološke proizvodnje vodika z cianobakterijam: pregled z biološkega vidika.]] Monika Škrjanc, 11. junija<br /> # Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant (M. Folcher; Nature Communications 5, 1–11, 2014; http://www.nature.com/ncomms/2014/141111/ncomms6392/full/ncomms6392.html) [[Z EEG nadzorovano izražanje transgena preko brezžično napajanega optogenetskega celičnega vsadka.]] Luka Smole, 11. junija 2015</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=MBT_seminarji_2015&diff=10659 2015-06-11T23:51:34Z

<p>Uros.Stupar: </p> <hr /> <div>'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2014/15'''<br /> <br /> Tabela za razpored po tednih bo objavljena v spletni učilnici, vanjo pa se vpišite tudi za kratke predstavitve novic (3 min, dvakrat v semestru). Na tej strani bo samo seznam odobrenih člankov za seminar in povezave do člankov in do povzetkov, ki jih morate objaviti najkasneje tri dni pred predstavitvijo (ponedeljek oz. torek). Angleški naslov prevedite tudi v slovenščino - to bo naslov povzetka, ki ga objavite na posebni strani, tako kot so to naredili kolegi pred vami (oz. lani).<br /> <br /> Način vnosa:<br /> <br /> # The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops &amp; Food 5(1), 2014; http://dx.doi.org/10.4161/gmcr.26979) Pomen glutation peroksidaze 8 iz repnjakovca za zaščito rastline ''Arabidopsis thaliana'' in bakterije ''Escherichia coli'' pred oksidativnim stresom. Janez Novak, 15. marca 2014<br /> (slovenski naslov povežite z novo stranjo, na kateri bo povzetek)<br /> <br /> <br /> '''Naslovi odobrenih člankov po temah:'''<br /> <br /> '''Gensko spremenjene rastline'''&lt;br&gt;<br /> # Successful high-level accumulation of fish oil omega-3 long-chain polyunsaturated fatty acids in a transgenic oilseed crop (Ruiz-Lopez, N., et al; The plant journal 77, 198-208, 2014; http://www.ncbi.nlm.nih.gov/pubmed/24308505). [[Uspešna priprava gensko spremenjene oljne rastline z visoko vsebnostjo omega-3 polinenasičenih maščobnih kislin.]] Petra Malavašič, 20. marca 2015<br /> #A simplified and accurate detection of the genetically modified wheat MON71800 with one calibrator plasmid (Jae Juan, S.,et al; Food Chemistry 176, 1-6, ;http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S03088146140196572015 [[Poenostavljena in točna detekcija gensko spemenjene pšenice MON71800 z enim kalibratorskim plazmidom]]. Matej Lesar, 20. marca 2015<br /> <br /> '''Gensko spremenjene živali'''&lt;br&gt;<br /> # [[A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expresion]] (H. Chen; et all.; BMC Biotechnology 2015, 15:4, doi:10.1186/s12896-015-0121-4; http://www.biomedcentral.com/1472-6750/15/4). Edvinas Grauželis, 27. marca 2015 (in English)<br /> # Production of functional active human growth factors in insects used as living biofactories (B. Dudognon, et al; Journal of Biotechnology 184, 229–239, 2014; http://dx.doi.org/10.1016/j.jbiotec.2014.05.030). [[Proizvodnja funkcionalno aktivnih človeških rastnih faktorjev v insektih uporabljenih kot žive biotovarne]] Maxi Sagmeister, 27. marca 2015<br /> <br /> '''Okolje'''&lt;br&gt;<br /> # Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles (Yuan Gao ''et al.'', Enzyme and Microbial Technology, vol. 54, pages 38-44, 10.1.2014, http://www.sciencedirect.com/science/article/pii/S0141022913002044). [[Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliestrski tekstil]]. Mitja Crček, 3. aprila 2015<br /> # Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene (A. W. Vail ''et al.''; Transgenic Research, 29. 11. 2014; http://link.springer.com/article/10.1007/s11248-014-9851-7). [[Biorazgradnja atrazina s tremi transgenskimi travami in lucerno, ki izražajo gen za modificirano bakterijsko atrazin klorohidrolazo]]. Mirjam Kmetič, 3. aprila 2015 <br /> <br /> '''Terapevtiki'''&lt;br&gt;<br /> # Glycosylated enfuvirtide: A long-lasting glycopeptide with potent anti-HIV activity; http://pubs.acs.org/doi/full/10.1021/jm5016582 [[Glikoliziran Enfuvirtid: glikopeptid z močno proti HIV aktivnostjo s podaljšanim delovanjem]]. Sebastian Pleško, 10. aprila <br /> # Microbicidal effects of α- and θ-defensins against antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa; http://ini.sagepub.com/content/21/1/17.long. [[Mikrobicidno delovanje α in θ defenzinov na antibiotik-odporne Staphylococcus aureus in Pseudomonas aeruginosa]]. Ana Kapraljević, 10. aprila<br /> <br /> '''Encimi'''&lt;br&gt;<br /> # Immobilization and controlled release of β-galactosidase from chitosan-grafted hydrogels; http://www.sciencedirect.com/science/article/pii/S0308814615001028. [[Imobilizacija in nadzorovano sproščanje β-galaktozidaze iz hitozanskega hidrogela]]. Mojca Banič, 16. aprila 2015<br /> # Construction of efficient xylose utilizing ''Pichia pastoris'' for industrial enzyme production (Li ''et al''; Microbial Cell Factories 14:22, 1-10, 2015; http://www.microbialcellfactories.com/content/14/1/22). [[Priprava Pichie pastoris, ki učinkovito uporablja ksilozo, za industrijsko proizvodnjo encimov]]. Špela Tomaž, 17. aprila 2015<br /> # Postharvest application of a novel chitinase cloned from ''Metschnikowia fructicola'' and overexpressed in ''Pichia pastoris'' to control brown rot of peaches; http://www.sciencedirect.com/science/article/pii/S0168160515000033. [[Uporaba hitinaze, klonirane iz Metschnikowie fructicola in prekomerno izražene v Pichii pastoris za nadzor rjave gnilobe breskev po obiranju]] Špela Pohleven, 17. aprila 2015<br /> <br /> '''Protitelesa'''&lt;br&gt; <br /> # Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells; http://dx.plos.org/10.1371/journal.pone.0116878. Optimizacija signalnih peptidov težkih in lahkih verig za večjo ekspresijo terapevtskih protiteles v CHO celičnih linijah. [[Optimizacija signalnih peptidov težkih in lahkih verig za večjo ekspresijo terapevtskih protiteles v CHO celičnih linijah]] Tjaša Blatnik, 23. aprila 2015<br /> # Ethanol precipitation for purification of recombinant antibodies (A. Tscheliessnig ''et al''; Journal of Biotechnology 188, 17-28, 2014; http://www.sciencedirect.com/science/article/pii/S0168165614007810). [[Čiščenje rekombinantnih protiteles z obarjanjem z etanolom]]. Urška Rauter, 24. aprila 2015<br /> # Functional mutations in and characterization of VHH against ''Helicobacter pylori'' urease (R. Hoseinpoor ''et al''; Applied Biochemistry and Biotechnology 172, 3079-3091, 2014; http://link.springer.com/article/10.1007/s12010-014-0750-4). [[Funkcionalne mutacije in karakterizacija VHH proti ureazi Helicobacter pylori]]. Marko Radojković, 7. maja 2015<br /> <br /> '''Cepiva'''&lt;br&gt;<br /> # Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions; http://www.sciencedirect.com/science/article/pii/S0378517315001507. [[Razvoj pegiliranih lipopleksov proti E6 za aplikacijo na sluznico pri predrakavih spremembah materničnega vratu]]. Tanja Korpar, 7. maja 2015<br /> # A novel “priming-boosting” strategy for immune interventions in cervical cancer (S. Liao et al.; Molecular Immunology 64, 295-305, 2015, http://www.sciencedirect.com/science/article/pii/S0161589014003460. [[Nova &quot;priming-boosting&quot; strategija za imunsko posredovanje pri raku materničnega vratu]]. Anita Kustec, 8. maja 2015<br /> # Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors (H.C. Wang et al.; Immunology letters 163, 206-213, 2015, http://www.ncbi.nlm.nih.gov/pubmed/25102364 ) [[Izboljšava cepiv proti antraksu z uporabo iz virusnih replikonov izvedenih vektorjev, ki omogočajo izražanje zaščitnega antigena.]] Daša Pavc, 8. maja 2015<br /> <br /> '''Male molekule in polimeri'''&lt;br&gt;<br /> # Methanol-induced chain termination in poly(3-hydroxybutyrate) biopolymers: Molecular weight control; http://www.sciencedirect.com/science/article/pii/S0141813014008307. [[Z metanolom inducirana terminacija polimerizacije poli(3-hidroksibutiratnih) polimerov: Vpliv na molekulsko maso]]. Gašper Lavrenčič, 14. maja 2015<br /> # Purification and characterization of gamma poly glutamic acid from newly Bacillus licheniformis NRC20; http://www.sciencedirect.com/science/article/pii/S0141813014008216. [[Čiščenje in karakterizacija gama poliglutaminske kisline iz bakterije Bacillus licheniformis NRC20]] Uroš Stupar, 14. maja 2015<br /> # Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases (Citorik RJ. ''et al''; Nature Biotechnology 32, 1141-1145, 2014; http://www.nature.com/nbt/journal/v32/n11/full/nbt.3011.html). [[Sekven%C4%8Dno specifi%C4%8Dna protimikrobna sredstva]] Iza Ogris, 15. maja 2015<br /> # Chromosomal integration of hyaluronic acid synthesis (''has'') genes enhances the molecular weight of hyaluronan produced in ''Lactococcus lactis'' (R. V. Hmar et al; Biotechnol. J. 9 (12), 2014; http://dx.doi.org/10.1002/biot.201400215) [[Integracija genov za sintezo hialuronske kisline v kromosom bakterije Lactococcus lactis izboljša sintezo visokomolekularne hialuronske kisline]] Maja Grdadolnik, 15. maja 2015<br /> <br /> '''Pretvorba biomase'''&lt;br&gt;<br /> # Effect of pretreatment methods on the synergism of cellulase and xylanase during the hydrolysis of bagasse (L. Jia ''et al''; Bioresource Technology 185, 2015; http://www.sciencedirect.com/science/article/pii/S0960852415002114) [[Vpliv metod predobdelave na sinergizem celulaze in ksilanaze pri hidrolizi bagase]]. Eva Lucija Kozak, 21. maja 2015<br /> # Third generation biohydrogen production by Clostridium butyricum and adapted mixed cultures from Scenedesmus obliquus microalga biomass; http://www.sciencedirect.com/science/article/pii/S0016236115002550?np=y [[Tretja generacija proizvodnje biovodika s pomočjo, z mikroalgami Scenedesmus obliquus hranjenimi bakterijami Clostridium butyricum in mešanico prilagojenih mikroorganizmov]] Nives Naraglav, 22. maja 2015<br /> # Bio-catalytic action of twin-screw extruder enzymatic hydrolysis on the deconstruction of annual plant material: Case of sweet corn co-products; http://www.sciencedirect.com/science/article/pii/S0926669015000436 [[Biokatalitični učinek encimske hidrolize dvovijačnega ekstruderja na destrukturiranje rastlinskega materiala]]. Griša Prinčič, 22. maja 2015<br /> <br /> '''Metabolično inženirstvo'''&lt;br&gt;<br /> # Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica (K. Qiao ''et al.''; Metabolic Engineering 29, 2014; http://www.sciencedirect.com/science/article/pii/S1096717615000166) [[Povečanje proizvodnje lipidov v kvasovki Yarrowia lipolytica]]. Andreja Bratovš, 28. maja 2015<br /> # Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals (Weerawat Runguphana, Jay D. Keasling; Metabolic Engineering, vol 21, January 2014, Pages 103–113; http://www.sciencedirect.com/science/article/pii/S1096717613000670). [[Metabolno inženirstvo kvasovke Saccharomyces cerevisiae za proizvodnjo biogoriva in kemikalij iz maščobnih kislin]]. Dominik Kert, 29. maja 2015<br /> # Metabolic engineering of Klebsiella pneumoniae for the production of cis,cis-muconic acid (Jung,H.-M. Jung,M.-Y. Oh, M.-K.;Applied Microbiology and Biotechnology, Published online: 14 February 2015; http://link.springer.com/article/10.1007/s00253-015-6442-3). [[Metabolno inženirstvo Klebsiella pneumoniae za produkcijo cis,cis-mukonične kisline]]. Jure Zabret, 29. maja 2015<br /> <br /> '''Biološki viri energije'''&lt;br&gt;<br /> # Anodic and cathodic microbial communities in single chamber microbial fuel cells; http://www.sciencedirect.com/science/article/pii/S1871678414021694. [[Anodna in katodna mikrobna združba v eno-celični mikrobni gorivni celici]] Tamara Marić, 4. junija 2015<br /> # Combination of dry dark fermentation and mechanical pretreatment for lignocellulosic deconstruction: An innovative strategy for biofuels and volatile fatty acids recovery; http://www.sciencedirect.com/science/article/pii/S0306261915002196. [[Kombinacija temne fermentacije v trdnem stanju in mehanske obdelave za razgradnjo lignoceluloze: Inovativen pristop za proizvodnjo biogoriv in hlapnih organskih kislin.]] Jernej Pušnik, 4. junija 2015<br /> # Potential use of feedlot cattle manure for bioethanol production; http://www.sciencedirect.com/science/article/pii/S0960852415001960. [[Uporaba govejega gnoja v proizvodnji bioetanola.]] Nastja Pirman, 5. junija 2015<br /> # Cellulolytic enzymes produced by a newly isolated soil fungus Penicillium sp. TG2 with potential for use in cellulosic ethanol production; http://www.sciencedirect.com/science/article/pii/S0960148114007022. [[Celulolitični encimi talne glive Penicillium sp. TG2 in njihov potencial pri proizvodnji etanola.]] Jana Verbančič, 5. junija 2015<br /> <br /> '''Novi pristopi v molekularni biotehnologiji'''&lt;br&gt;<br /> # Exploring the potential of algae/bacteria interactions; http://www.sciencedirect.com/science/article/pii/S0958166915000269. [[Potenciali interakcij med algami in bakterijami.]] Matja Zalar, 11. junija<br /> # How close we are to achieving commercially viable large-scale photobiological hydrogen production by cyanobacteria: A review of the biological aspects; http://www.mdpi.com/2075-1729/5/1/997/htm. [[Kako blizu smo dosegu komercialno dostopne masovne fotobiološke proizvodnje vodika z cianobakterijam: pregled z biološkega vidika.]] Monika Škrjanc, 11. junija<br /> # Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant (M. Folcher; Nature Communications 5, 1–11, 2014; http://www.nature.com/ncomms/2014/141111/ncomms6392/full/ncomms6392.html) [[Z EEG nadzorovano izražanje transgena preko brezžično napajanega optogenetskega celičnega vsadka.]] Luka Smole, 11. junija 2015</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10079 2015-01-20T08:16:48Z

<p>Uros.Stupar: /* Conclusion */</p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang. [http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, biotechnology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed&lt;ref name=&quot;ref1&quot;&gt;M. Jinek, A. East, A. Cheng, S. Lin, E. Ma, and J. Doudna. RNA-programmed genome editing in human cells. Elife, vol. 2013, pp. 1–9, 2013.&lt;/ref&gt;.<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone&lt;ref name=&quot;ref2&quot;&gt;Y. Fu, J. a Foden, C. Khayter, M. L. Maeder, D. Reyon, J. K. Joung, and J. D. Sander. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol., vol. 31, no. June, pp. 822–6, 2013.&lt;/ref&gt;&lt;ref name=&quot;ref3&quot;&gt;S. W. Cho, S. Kim, J. M. Kim, and J.-S. Kim. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol., vol. 31, no. 3, pp. 230–2, 2013.&lt;/ref&gt;.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. In NHEJ, the free double-stranded ends are bound by a heterodimer of two proteins, Ku70 and Ku 80, T hese proteins stabilize the ends and mark them for subsequent manipulations. The mechanisms that are not yet well understood, but we know the Ku70/S0 heterodimers act as handles used by other proteins to draw the two double-stranded ends close together so that enzymes can seal the break. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions&lt;ref name=&quot;ref4&quot;&gt;S. Burma, B. P. C. Chen, and D. J. Chen. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst)., vol. 5, pp. 1042–1048, 2006.&lt;/ref&gt;.<br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α) promoter. Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need&lt;ref name=&quot;ref5&quot;&gt;A. J. Pierce, J. M. Stark, F. D. Araujo, M. E. Moynahan, M. Berwick, and M. Jasin. Double-strand breaks and tumorigenesis. Trends Cell Biol., vol. 11, no. ll, pp. 52–59, 2001.&lt;/ref&gt;.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems. These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific.&lt;ref name=&quot;ref6&quot;&gt;M. Baker. Gene editing at CRISPR speed. Nat. Biotechnol., vol. 32, no. 4, pp. 309–12, 2014.&lt;/ref&gt; Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.<br /> <br /> ==References==<br /> <br /> &lt;references /&gt;<br /> <br /> [[SB students resources]]</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10078 2015-01-20T07:56:04Z

<p>Uros.Stupar: </p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang. [http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, biotechnology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed&lt;ref name=&quot;ref1&quot;&gt;M. Jinek, A. East, A. Cheng, S. Lin, E. Ma, and J. Doudna. RNA-programmed genome editing in human cells. Elife, vol. 2013, pp. 1–9, 2013.&lt;/ref&gt;.<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone&lt;ref name=&quot;ref2&quot;&gt;Y. Fu, J. a Foden, C. Khayter, M. L. Maeder, D. Reyon, J. K. Joung, and J. D. Sander. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol., vol. 31, no. June, pp. 822–6, 2013.&lt;/ref&gt;&lt;ref name=&quot;ref3&quot;&gt;S. W. Cho, S. Kim, J. M. Kim, and J.-S. Kim. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol., vol. 31, no. 3, pp. 230–2, 2013.&lt;/ref&gt;.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. In NHEJ, the free double-stranded ends are bound by a heterodimer of two proteins, Ku70 and Ku 80, T hese proteins stabilize the ends and mark them for subsequent manipulations. The mechanisms that are not yet well understood, but we know the Ku70/S0 heterodimers act as handles used by other proteins to draw the two double-stranded ends close together so that enzymes can seal the break. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions&lt;ref name=&quot;ref4&quot;&gt;S. Burma, B. P. C. Chen, and D. J. Chen. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst)., vol. 5, pp. 1042–1048, 2006.&lt;/ref&gt;.<br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α) promoter. Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need&lt;ref name=&quot;ref5&quot;&gt;A. J. Pierce, J. M. Stark, F. D. Araujo, M. E. Moynahan, M. Berwick, and M. Jasin. Double-strand breaks and tumorigenesis. Trends Cell Biol., vol. 11, no. ll, pp. 52–59, 2001.&lt;/ref&gt;.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific.&lt;ref name=&quot;ref6&quot;&gt;M. Baker. Gene editing at CRISPR speed. Nat. Biotechnol., vol. 32, no. 4, pp. 309–12, 2014.&lt;/ref&gt; Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.<br /> <br /> ==References==<br /> <br /> &lt;references /&gt;<br /> <br /> [[SB students resources]]</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10077 2015-01-20T07:55:54Z

<p>Uros.Stupar: </p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang.[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, biotechnology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed&lt;ref name=&quot;ref1&quot;&gt;M. Jinek, A. East, A. Cheng, S. Lin, E. Ma, and J. Doudna. RNA-programmed genome editing in human cells. Elife, vol. 2013, pp. 1–9, 2013.&lt;/ref&gt;.<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone&lt;ref name=&quot;ref2&quot;&gt;Y. Fu, J. a Foden, C. Khayter, M. L. Maeder, D. Reyon, J. K. Joung, and J. D. Sander. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol., vol. 31, no. June, pp. 822–6, 2013.&lt;/ref&gt;&lt;ref name=&quot;ref3&quot;&gt;S. W. Cho, S. Kim, J. M. Kim, and J.-S. Kim. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol., vol. 31, no. 3, pp. 230–2, 2013.&lt;/ref&gt;.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. In NHEJ, the free double-stranded ends are bound by a heterodimer of two proteins, Ku70 and Ku 80, T hese proteins stabilize the ends and mark them for subsequent manipulations. The mechanisms that are not yet well understood, but we know the Ku70/S0 heterodimers act as handles used by other proteins to draw the two double-stranded ends close together so that enzymes can seal the break. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions&lt;ref name=&quot;ref4&quot;&gt;S. Burma, B. P. C. Chen, and D. J. Chen. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst)., vol. 5, pp. 1042–1048, 2006.&lt;/ref&gt;.<br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α) promoter. Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need&lt;ref name=&quot;ref5&quot;&gt;A. J. Pierce, J. M. Stark, F. D. Araujo, M. E. Moynahan, M. Berwick, and M. Jasin. Double-strand breaks and tumorigenesis. Trends Cell Biol., vol. 11, no. ll, pp. 52–59, 2001.&lt;/ref&gt;.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific.&lt;ref name=&quot;ref6&quot;&gt;M. Baker. Gene editing at CRISPR speed. Nat. Biotechnol., vol. 32, no. 4, pp. 309–12, 2014.&lt;/ref&gt; Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.<br /> <br /> ==References==<br /> <br /> &lt;references /&gt;<br /> <br /> [[SB students resources]]</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10076 2015-01-20T07:54:44Z

<p>Uros.Stupar: </p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang,[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, biotechnology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed&lt;ref name=&quot;ref1&quot;&gt;M. Jinek, A. East, A. Cheng, S. Lin, E. Ma, and J. Doudna. RNA-programmed genome editing in human cells. Elife, vol. 2013, pp. 1–9, 2013.&lt;/ref&gt;.<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone&lt;ref name=&quot;ref2&quot;&gt;Y. Fu, J. a Foden, C. Khayter, M. L. Maeder, D. Reyon, J. K. Joung, and J. D. Sander. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol., vol. 31, no. June, pp. 822–6, 2013.&lt;/ref&gt;&lt;ref name=&quot;ref3&quot;&gt;S. W. Cho, S. Kim, J. M. Kim, and J.-S. Kim. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol., vol. 31, no. 3, pp. 230–2, 2013.&lt;/ref&gt;.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. In NHEJ, the free double-stranded ends are bound by a heterodimer of two proteins, Ku70 and Ku 80, T hese proteins stabilize the ends and mark them for subsequent manipulations. The mechanisms that are not yet well understood, but we know the Ku70/S0 heterodimers act as handles used by other proteins to draw the two double-stranded ends close together so that enzymes can seal the break. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions&lt;ref name=&quot;ref4&quot;&gt;S. Burma, B. P. C. Chen, and D. J. Chen. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst)., vol. 5, pp. 1042–1048, 2006.&lt;/ref&gt;.<br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α) promoter. Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need&lt;ref name=&quot;ref5&quot;&gt;A. J. Pierce, J. M. Stark, F. D. Araujo, M. E. Moynahan, M. Berwick, and M. Jasin. Double-strand breaks and tumorigenesis. Trends Cell Biol., vol. 11, no. ll, pp. 52–59, 2001.&lt;/ref&gt;.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific.&lt;ref name=&quot;ref6&quot;&gt;M. Baker. Gene editing at CRISPR speed. Nat. Biotechnol., vol. 32, no. 4, pp. 309–12, 2014.&lt;/ref&gt; Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.<br /> <br /> ==References==<br /> <br /> &lt;references /&gt;<br /> <br /> [[SB students resources]]</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10075 2015-01-20T06:33:51Z

<p>Uros.Stupar: /* NHEJ (Non-homologous end joining) */</p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang,[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, biotechnology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed&lt;ref name=&quot;ref1&quot;&gt;M. Jinek, A. East, A. Cheng, S. Lin, E. Ma, and J. Doudna. RNA-programmed genome editing in human cells. Elife, vol. 2013, pp. 1–9, 2013.&lt;/ref&gt;.<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone&lt;ref name=&quot;ref2&quot;&gt;Y. Fu, J. a Foden, C. Khayter, M. L. Maeder, D. Reyon, J. K. Joung, and J. D. Sander. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol., vol. 31, no. June, pp. 822–6, 2013.&lt;/ref&gt;&lt;ref name=&quot;ref3&quot;&gt;S. W. Cho, S. Kim, J. M. Kim, and J.-S. Kim. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol., vol. 31, no. 3, pp. 230–2, 2013.&lt;/ref&gt;.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. In NHEJ, the free double-stranded ends are bound by a heterodimer of two proteins, Ku70 and Ku 80, T hese proteins stabilize the ends and mark them for subsequent manipulations. The mechanisms that are not yet well understood, but we know the Ku70/S0 heterodimers act as handles used by other proteins to draw the two double-stranded ends close together so that enzymes can seal the break. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions&lt;ref name=&quot;ref4&quot;&gt;S. Burma, B. P. C. Chen, and D. J. Chen. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst)., vol. 5, pp. 1042–1048, 2006.&lt;/ref&gt;.<br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need&lt;ref name=&quot;ref5&quot;&gt;A. J. Pierce, J. M. Stark, F. D. Araujo, M. E. Moynahan, M. Berwick, and M. Jasin. Double-strand breaks and tumorigenesis. Trends Cell Biol., vol. 11, no. ll, pp. 52–59, 2001.&lt;/ref&gt;.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific.&lt;ref name=&quot;ref6&quot;&gt;M. Baker. Gene editing at CRISPR speed. Nat. Biotechnol., vol. 32, no. 4, pp. 309–12, 2014.&lt;/ref&gt; Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.<br /> <br /> ==References==<br /> <br /> &lt;references /&gt;<br /> <br /> [[SB students resources]]</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10052 2015-01-19T23:52:35Z

<p>Uros.Stupar: </p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang,[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, biotechnology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed&lt;ref name=&quot;ref1&quot;&gt;M. Jinek, A. East, A. Cheng, S. Lin, E. Ma, and J. Doudna. RNA-programmed genome editing in human cells. Elife, vol. 2013, pp. 1–9, 2013.&lt;/ref&gt;.<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone&lt;ref name=&quot;ref2&quot;&gt;Y. Fu, J. a Foden, C. Khayter, M. L. Maeder, D. Reyon, J. K. Joung, and J. D. Sander. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol., vol. 31, no. June, pp. 822–6, 2013.&lt;/ref&gt;&lt;ref name=&quot;ref3&quot;&gt;S. W. Cho, S. Kim, J. M. Kim, and J.-S. Kim. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol., vol. 31, no. 3, pp. 230–2, 2013.&lt;/ref&gt;.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions&lt;ref name=&quot;ref4&quot;&gt;S. Burma, B. P. C. Chen, and D. J. Chen. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst)., vol. 5, pp. 1042–1048, 2006.&lt;/ref&gt;. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need&lt;ref name=&quot;ref5&quot;&gt;A. J. Pierce, J. M. Stark, F. D. Araujo, M. E. Moynahan, M. Berwick, and M. Jasin. Double-strand breaks and tumorigenesis. Trends Cell Biol., vol. 11, no. ll, pp. 52–59, 2001.&lt;/ref&gt;.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific.&lt;ref name=&quot;ref6&quot;&gt;M. Baker. Gene editing at CRISPR speed. Nat. Biotechnol., vol. 32, no. 4, pp. 309–12, 2014.&lt;/ref&gt; Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.<br /> <br /> ==References==<br /> <br /> &lt;references /&gt;<br /> <br /> [[SB students resources]]</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10044 2015-01-19T23:47:24Z

<p>Uros.Stupar: </p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang,[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, biotechnology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed&lt;ref name=&quot;ref1&quot;&gt;M. Jinek, A. East, A. Cheng, S. Lin, E. Ma, and J. Doudna. RNA-programmed genome editing in human cells. Elife, vol. 2013, pp. 1–9, 2013.&lt;/ref&gt;.<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone&lt;ref name=&quot;ref2&quot;&gt;Y. Fu, J. a Foden, C. Khayter, M. L. Maeder, D. Reyon, J. K. Joung, and J. D. Sander. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol., vol. 31, no. June, pp. 822–6, 2013.&lt;/ref&gt;&lt;ref name=&quot;ref3&quot;&gt;S. W. Cho, S. Kim, J. M. Kim, and J.-S. Kim. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol., vol. 31, no. 3, pp. 230–2, 2013.&lt;/ref&gt;.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions&lt;ref name=&quot;ref4&quot;&gt;S. Burma, B. P. C. Chen, and D. J. Chen. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst)., vol. 5, pp. 1042–1048, 2006.&lt;/ref&gt;. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific.&lt;ref name=&quot;ref5&quot;&gt;M. Baker. Gene editing at CRISPR speed. Nat. Biotechnol., vol. 32, no. 4, pp. 309–12, 2014.&lt;/ref&gt; Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.<br /> <br /> ==References==<br /> <br /> &lt;references /&gt;<br /> <br /> [[SB students resources]]</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10041 2015-01-19T23:43:33Z

<p>Uros.Stupar: </p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang,[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, biotechnology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed&lt;ref name=&quot;ref1&quot;&gt;M. Jinek, A. East, A. Cheng, S. Lin, E. Ma, and J. Doudna. RNA-programmed genome editing in human cells. Elife, vol. 2013, pp. 1–9, 2013.&lt;/ref&gt;.<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone&lt;ref name=&quot;ref2&quot;&gt;Y. Fu, J. a Foden, C. Khayter, M. L. Maeder, D. Reyon, J. K. Joung, and J. D. Sander. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol., vol. 31, no. June, pp. 822–6, 2013.&lt;/ref&gt;&lt;ref name=&quot;ref3&quot;&gt;S. W. Cho, S. Kim, J. M. Kim, and J.-S. Kim. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol., vol. 31, no. 3, pp. 230–2, 2013.&lt;/ref&gt;.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions&lt;ref name=&quot;ref4&quot;&gt;S. Burma, B. P. C. Chen, and D. J. Chen. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst)., vol. 5, pp. 1042–1048, 2006.&lt;/ref&gt;. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific. Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.<br /> <br /> ==References==<br /> <br /> &lt;references /&gt;<br /> <br /> [[SB students resources]]</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10038 2015-01-19T23:33:04Z

<p>Uros.Stupar: </p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang,[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, biotechnology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone&lt;ref name=&quot;ref1&quot;&gt;M. Jinek, A. East, A. Cheng, S. Lin, E. Ma, and J. Doudna. RNA-programmed genome editing in human cells. Elife, vol. 2013, pp. 1–9, 2013.&lt;/ref&gt;.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions&lt;ref name=&quot;ref2&quot;&gt;S. Burma, B. P. C. Chen, and D. J. Chen. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst)., vol. 5, pp. 1042–1048, 2006.&lt;/ref&gt;. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific. Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.<br /> <br /> ==References==<br /> <br /> &lt;references /&gt;<br /> <br /> [[SB students resources]]</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10037 2015-01-19T23:31:58Z

<p>Uros.Stupar: </p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang,[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, biotechnology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone&lt;ref name=&quot;ref2&quot;&gt;M. Jinek, A. East, A. Cheng, S. Lin, E. Ma, and J. Doudna. RNA-programmed genome editing in human cells. Elife, vol. 2013, pp. 1–9, 2013.&lt;/ref&gt;.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions&lt;ref name=&quot;ref2&quot;&gt;S. Burma, B. P. C. Chen, and D. J. Chen. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst)., vol. 5, pp. 1042–1048, 2006.&lt;/ref&gt;. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific. Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.<br /> <br /> ==References==<br /> <br /> &lt;references /&gt;<br /> <br /> [[SB students resources]]</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10036 2015-01-19T23:27:08Z

<p>Uros.Stupar: </p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang,[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, biotechnology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). <br /> <br /> This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. <br /> But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions&lt;ref name=&quot;ref2&quot;&gt;S. Burma, B. P. C. Chen, and D. J. Chen. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst)., vol. 5, pp. 1042–1048, 2006.&lt;/ref&gt;. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific. Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.<br /> <br /> ==References==<br /> <br /> &lt;references /&gt;<br /> <br /> [[SB students resources]]</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10035 2015-01-19T23:26:34Z

<p>Uros.Stupar: </p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang,[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, biotechnology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). <br /> <br /> This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. <br /> But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions&lt;ref name=&quot;ref2&quot;&gt;S. Burma, B. P. C. Chen, and D. J. Chen. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst)., vol. 5, pp. 1042–1048, 2006.&lt;/ref. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific. Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.<br /> <br /> ==References==<br /> <br /> &lt;references /&gt;<br /> <br /> [[SB students resources]]</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10034 2015-01-19T23:21:46Z

<p>Uros.Stupar: </p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang,[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, biotechnology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). <br /> <br /> This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. <br /> But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific. Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.<br /> <br /> ==References==<br /> <br /> &lt;references /&gt;<br /> <br /> [[SB students resources]]</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10033 2015-01-19T23:18:27Z

<p>Uros.Stupar: /* Introduction */</p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang,[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, biotechnology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). <br /> <br /> This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. <br /> But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific. Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10032 2015-01-19T23:18:03Z

<p>Uros.Stupar: /* Introduction */</p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang,[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an valuable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, bio¬technology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). <br /> <br /> This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. <br /> But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific. Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10031 2015-01-19T23:17:13Z

<p>Uros.Stupar: </p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang,[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> <br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an invaluable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, bio¬technology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). <br /> <br /> This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. <br /> But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific. Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10030 2015-01-19T23:17:03Z

<p>Uros.Stupar: </p> <hr /> <div>L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. a Marraffini, and F. Zhang,[http://zlab.mit.edu/assets/reprints/Cong_L_Science_2013.pdf Multiplex genome engineering using CRISPR/Cas systems]. Science, vol. 339, pp. 819–23, 2013.<br /> Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an invaluable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, bio¬technology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). <br /> <br /> This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. <br /> But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific. Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10029 2015-01-19T23:00:13Z

<p>Uros.Stupar: </p> <hr /> <div>Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an invaluable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, bio¬technology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). <br /> <br /> This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. <br /> But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> <br /> ==Multiplex genome engineering using a single CRISPR array==<br /> <br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific. Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10028 2015-01-19T22:59:06Z

<p>Uros.Stupar: </p> <hr /> <div>Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an invaluable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, bio¬technology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). <br /> <br /> This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. <br /> But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone.<br /> <br /> <br /> <br /> ==NHEJ (Non-homologous end joining)==<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> Multiplex genome engineering using a single CRISPR array<br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific. Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Multiplex_genome_engineering_using_CRISPR/Cas_systems&diff=10027 2015-01-19T22:58:41Z

<p>Uros.Stupar: New page: Uroš Stupar ==Introduction== Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyr...</p> <hr /> <div>Uroš Stupar<br /> <br /> ==Introduction==<br /> <br /> Here I will try to describe you how type II procaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system from Streptococcus pyrogenes can be efficiently used to precisely edit genome sequences. In this article the scientists described the engineering of two different type II CRISPR/Cas adaptive immune systems to induce cleavage by enzyme Cas9 guided by short RNA segments, in a determined genomic loci in both human and mouse cells. They also converted Cas9 into a nicking enzyme that helps DNA repair. And last but not least, they encoded a variety of guide sequences in a CRISPR array, so CRISPR/Cas9 system could edit many sites within a genome at the same time.<br /> <br /> Besides serving as an invaluable research tool, targeted genome engineering in cells and organisms could potentially provide the path to revolutionary applications in medicinal human therapies, molecular biology, bio¬technology and microbial engineering. Methods of modifying the genome exploit endogenous DNA repair pathways that are initiated by the introduction of site-specific double-stranded DNA cleavages. Induction of site-specific genomic mutations in cell cultures was made possible with new methods that include zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALEs). These enzymes target and induce cleavages in specific genomic DNA sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes, which requires a lot of time and hard work. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. It is still time consuming. We needed new technologies that are affordable and easy to engineer. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed<br /> <br /> ==CRISPR/Cas system== <br /> <br /> The type II CRISPR/Cas9 system is used by bacteria as an RNA-guided defense system against invading viruses and plasmids. The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes. One of these genes is Cas9 nuclease and the others are two noncoding CRISPRR RNAs (crRNA), a trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) which contains spacers (nuclease guide sequences). Between the spacers there are identical direct repeats. In Streptococcus pyogenes the spacers make base pairs by noncovalent hydrogen bonding (hybridization) with complementary target DNA sequence. The Cas9 endonuclease is then guided to the target site with the help of tracrRNA, which recruits crRNA into the Cas9 complex. Cas9 then induces double stranded breaks in the targeted DNA. This system was developed throughout evolution to cleave foreign DNA (viral DNA for example). <br /> <br /> This system can be modified and adapted for inducing site-specific genome mutations and thus edit target DNA sequences. The mature crRNA and tracrRNA can be fused in a single synthetic guide RNA (sgRNA). The Cas9 is then recruited by crRNA and tracrRNA, which bind to the target site, where double-strand breaks are induced with its catalytic activity. In previous studies it has been shown that tracrRNA, pre-crRNA, RNase III and Cas9 nuclease are sufficient to induce double-stranded DNA breaks in vitro and in prokaryotics cells. <br /> But the cells try to repair the damaged RNA through a variety of mechanisms. NHEJ (Non-homologous end joining) is one of the mechanisms most commonly used by cells to repair double stranded breaks in DNA backbone.<br /> <br /> <br /> <br /> NHEJ (Non-homologous end joining)<br /> <br /> Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA It is referred to as non-homologous because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. This term was first used by Moore and Haber in 1996. NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately. Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, which are present in tumor cells. NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells. The choice between NHEJ and homologous recombination for repair of a double-strand break is regulated at the initial step in recombination, 5' end resection. In this step, the 5' strand of the break is degraded by nucleases to create long 3' single-stranded tails. DSBs that have not been resected can be rejoined by NHEJ, but resection of even a few nucleotides strongly inhibits NHEJ and effectively commits the break to repair by recombination. NHEJ is active throughout the cell cycle, but is most important during G1 when no homologous template for recombination is available. This regulation is accomplished by the cyclin-dependent kinase Cdk1 (Cdc28 in yeast), which is turned off in G1 and expressed in S and G2. Cdk1 phosphorylates the nuclease Sae2, allowing NHEJ to initiate. So when the double-strand breaks are induced in the target DNA sequence by Cas9 complex, cell mechanisms try to repair those using NHEJ pathways. Because of the inaccuracy of the repair by NHEJ, the repaired DNA sequence can result in mutations at target sites such as small insertions or deletions. <br /> <br /> <br /> ==Effectivenes of the CRISPR/Cas system for targeted cleavage of mammalian chromosomes==<br /> <br /> In this study the nuclear localization signals were attached to optimized Cas9 and RNase III from Streptococcus pyogenes, because previous research showed this helps transfer these two enzymes in the nucleus where genomic DNA, which is going to be edited, is located. In another study GFP and mCherry coding sequences were attached to follow the localization of the Cas9 nuclease and RNase III in vivo (see Figure 1A). Expression of human codon–optimized Cas9 (hSpCas9) and RNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1α). Gene for tracrRNA was transferred under RNase III U6 promoter, as well as the pre-crRNA, which contained a single nucleus guiding sequence, flanked by two identical direct repeats. The nucleus guiding sequence was carefully designed so it binds a 30 base-pair long target sequence called protospacer, which is in our case located in human EMX1 gene (see Figure 1B). This gene encodes a member of the EMX family of transcriptional factors. The EMX1 gene, along with its family members, is expressed in the developing cerebrum (also known as the telencephalon). Emx1 plays a role in specification of positional identity, the proliferation of neural stem cells, differentiation of layer-specific neuronal phenotypes and commitment to aneuronal or glial cell fate. After the protospacer there are of three nucleotides – NGG. This is called protospacer-adjacent motif (PAM) and is necessary for the successful binding of the spacer.<br /> <br /> The components of this modified Streptococcus pyogenes CRISPR/Cas system were transfected in 293FT cells in different combinations, to test if the cleavage of DNA strands can be successfully achieved. In figure 4C we can see how base pairing between crRNA and target sequence should occur. Cleavage site is shown by the red arrow (see Figure 1C). For detection, the SURVEYOR assay was used. SURVEYOR Mutation Detection Kits are a simple and robust method to detect mutations and polymorphisms in DNA. The key component of the kits is Surveyor Nuclease, a member of the CEL family of mismatch-specific nucleases. Surveyor Nuclease recognizes and cleaves mismatches due to the presence of single nucleotide polymorphisms (SNPs) or small insertions or deletions. To be sure that the results are right, the samples were sequenced using Sanger sequencing. The results showed that with combination of all four components (Cas9, RNase III, tracrRNA and pre-crRNA) most efficient cleavage in protospacer was obtained, although RNase II was not necessary for the induction of double-stranded breaks (see Figure 1D). It seems that tracrRNA and pre-crRNA can be processed in its absence. They are supposing that in mammalian cells there are already some RNases that help with the maturation of these two RNAs. If they removed any of the other three components, the cleavage was not successful, so they came to a conclusion that a system made of a minimum of three components – Cas9, tracrRNA and pre-crRNA – can be successfully used to induce double stranded breaks in a DNA molecule. This was the first step of this research.<br /> <br /> ==Generalizability of RNA-guided genome editing in eukaryotic cells==<br /> <br /> In the next part of the research they encoded Cas9, tracrRNA and pre-crRNA on the same specifically designed vector. They also made chimeric DNA hybrids, which they encoded in vector instead of tracrRNA and pre-crRNA individually. This hybrid type was a little modified chimeric crRNA-tracrRNA hybrid, where processed and mature crRNA, which contains the guide sequence, was fused to only a part of tracrRNA across a synthetic stem loop to form a mature crRNA-tracrRNA duplex (see Figure 2B). The purpose of this test was to compare the effectiveness of pre-crRNA and tracrRNA with the effectiveness of the chimeric crRNA-tracrRNA duplex, but also to find out if this CRISPR/Cas system could target the other protospacers in the EMX1 loci too. They chose five protospacers on EMX1 locus (see Figure 2A). The results showed that not all designed RNAs could cleave the targeted sequences. So they decided to target additional sequences in other genes such as the human PVALB gene and mouse Th gene. As for the EMX1 gene the first thing to do was to design the correct pre-crRNAs and chimeric DNA duplexes to target those sequences. The results were similar. Using the correct pre-crRNA which then forms crRNA-tracrRNA duplex proved to be the most efficient method as the double strand breaks were identified in all three analyzed Th mouse genes and in one out of two human PVALB targets. As we can clearly see from the electrophoresis gel in figure 2C, efficiency of cleavage in the same DNA targets using chimeric RNA was either lower or undetectable (see Figure 2C). This probably occurred because the chimeric RNA is less stable or its expression is lower. It could be that the endogenous RNA interference machinery degraded chimeric RNA. One of options is also that the chimeric DNA was just not efficient enough in recruiting Cas9 or recognizing specific targets because of its secondary structures. <br /> <br /> ==Specificity of the CRISP/Cas system for targeted cleavage== <br /> <br /> Next they analyzed the connection between the mismatches in the guide sequence region and the protospacer, because cleavage must not be only efficient, but also specific (see Figure 3A). They found out that the presence of a single-base mismatch up to 11 base pairs on the 5’ direction of the PAM sequence, which is located immediately at the end of the guide sequence 3’ end, results in non-cleavage of the protospacer by Cas9. However mutations further upstream than 11 base pairs do not abolish the cleaving activity of the Cas9 nuclease (see Figure 3B). Similar results were obtained in previous bacterial and in vitro studies and now those results were confirmed. <br /> <br /> ==Targeted modification of genomes==<br /> <br /> Double stranded breaks in DNA sequences induced by type II CRISPR/Cas9 system can be repaired through different mechanisms. These mechanisms are crucial for preventing lethal DNA damage that can occur in cells and developed through the evolution. There are two main mechanisms. First one is homology directed repair (HDR), which can be used when there is a homologue piece of DNA present in the nucleus. It occurs mostly in G2 and S phase of the cell cycle. HDR is important for suppressing the formation of cancer. HDR maintains the genomic stability by repairing the broken DNA strand, assumed error free because of the use of a template. However when the homologue DNA piece is not available, another process called non-homologous end joining (NHEJ) can take place instead. When a double strand DNA lesion is repaired by NHEJ there is no validating DNA template present which may result in a non-original DNA strand formation with loss of information. A different nucleotide sequence in the DNA strand results in a different protein expressed in the cell. This protein may malfunction and processes in the cell may fail or take a different path. But in our case, we want to induce mutations, so this error-prone NHEJ mechanism is just what we need.<br /> <br /> Wild-type Cas9 induces site specific double strand breaks in DNA. In the study a mutation was induced in the RuvC I domain of Cas9 to convert an aspartate into an alanine (see Figure 4A). With this modification the enzyme becomes a DNA nickase (Cas9n) as only one DNA strand is cut by the enzyme. Nicked DNA is usually repaired through high-fidelity homology-directed repair, which means mutations rarely occur. 327 amplicons were then nicked by this modified Cas9n nickase and analyzed via SURVEYOR assay and Sanger sequencing. No deletions or insertions were detected (see Figure 4B). However in some rare cases nicked DNA can be also repaired through intermediates with double stranded breaks and NHEJ mechanism as shown in a previous research. They wanted to test also the Cas9 mediated homology directed repair at the EMX1 locus. To do this a homology repair template was added so the breaks in DNA would repair in the way the restriction sites for HindIII and NdeI restriction enzymes would be induced near the protospacer (see Figure 4C). Induction of the restriction sites was successful in both cases, using Cas9 and Cas9n. This was confirmed by digestion with HindIII restriction enzyme and electrophoresis separation of restriction fragments. In both cases they got a 2281 base pair long fragment, which is the non-restricted fragment and two fragments long 1189 and 1092 base pairs. The two smaller fragments show that the induction was successful in both Cas9 and Cas9n mediated HDR in approximately same percent (see Figure 4D). Just to be sure, the results were once more verified using Sanger sequencing. The advantage of the nickase is that it might reduce off-target mutations.<br /> Multiplex genome engineering using a single CRISPR array<br /> As described before the sequence of pre-crRNA in the CRISP locus contains spacers, which bind to a target DNA sequence between identical direct repeats. There can be one spacer, but there can be also multiple spacers for targeting multiple genes. In the end the scientists engineered a CRISPR array that contains two spacers for targeting EMX1 locus and PVLB locus. Efficient cleavage was detected in both (see Figure 4F). In figure 4F the design of crRNA is shown. Effectiveness of cleavage was tested using gel electrophoresis. As we can see from the gel, both protospacers were successfully cleaved.<br /> <br /> ==Conclusion==<br /> <br /> Type IICRISPR/Cas system was therefore shown as a perspective relatively new potential method for inducing RNA guided mutations by cleaving both strands of DNA molecule in target sites and relying on the error-prone NHEJ mechanism to induce mutations when repairing the DNA. A lot of research tool companies have already launched products for CRISPR-Cas systems65 These generally involve web-based bioinformatic tools to design gRNAs, plus RNA or plasmid vectors that encode Cas9, along with fluorescent proteins or other expression markers. Although this method is effective, specific and can target multiple targets, it can still be improved to even further to increase its effectivity. Like mentioned before it can only target sequences that are found in genome right next to three-nucleotide PAM sequences, which consist of NGG nucleotides. These repeats are only found once per 8 base pairs, which means not every part of sequence can serve as a protospacer. Furthermore complex secondary structures of crRNA and DNA metilation states can reduce the possibility of binding of guide sequences to the accessible protospacer. We should therefore explore further the family of Cas9 nucleases to find or engineer systems with better crRNA secondary structures for easier access to target sequences and different PAM requirements, so we can extend the choice of protospacers we can use for inducing mutagenesis. Specificity could also be improved. It could be improved with systems requiring multiple crRNA-Cas9 complexes for activity, reducing activity while increasing cooperativity and most importantly by a wise choice of guide sequences in crRNA. Also Cas9 proteins from species with larger genome than Streptococcus pyrogenes could be more specific. Despite all the improvements that need to be made in future, CRISPR/Cas system is cheaper and less time consuming than other methods for genome engineering that include zinc finger nucleases and TALE nucleases and seems to have all the potential to become a powerful tool in the future researches in the field of molecular biology, biotechnology and medicine.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=SB_students_resources&diff=10024 2015-01-19T22:52:08Z

<p>Uros.Stupar: </p> <hr /> <div>===Introduction to our students resources in Synthetic Biology===<br /> (Marko Dolinar)<br /> <br /> Synthetic biology made a vast progress in good 10 years since it established itself as an interdisciplinary field of research on the interface of molecular biology and engineering. University of Ljubljana Faculty of Chemistry and Chemical Technology has introduced a Synthetic Biology course as a part od Biochemistry MSc programme only in 2013/14. This is relatively late, considering a great success of Slovenian students at iGEM competitions since their first attendance in 2006. On the other hand, the field is still in its first stages if development and a complete textbook for a MSc level course is still missing. This is the reason why our students collaborated on the preparation of a Synthetic Biology textbook with the working title Synthetic Biology - A Students Textbook. It exists as a draft that is not publicly available and is actually part 1 of a (to be) 2-volumes title. Part I is subtitled Engineering Biology, while Part II (that currently doesn't exisist yet) will be subtitled Synthetic Biology Applications.<br /> <br /> As in all highly competitive fields of science and technology, students should be following recent progress by reading articles in high quality journals. However, this is often a very difficult task, especially at the BSc level. Specificities of the scientific and technical language, push of publishers towards very short methodological chapters and limited knowledge studens might have about advanced techniques make understanding papers a very challenging task. Therefore, I decided to face MSc students with the challenge to explain selected SB articles in a manner that would make the content of these articles understandable to BSc level students and non-experts.<br /> <br /> In 2014/15, seminars in Synthetic Biology include explanations and presentations of some of the top-cited articles from the field of Synthetic Biology. I compiled a list of 95 articles published between 2000 and 2014 having the highest number of citations according to the Web of Science database. The list ended with the paper just exceeding the 100 citations limit. Not included in the list were reviews. With 20 students enrolled in the course, the list has been further reduced to top 40 papers in the field. Students have been asked to check for content (they further eliminated 3 papers which proved to be reviews) and availabitly (they all seemed to be available as full texts with our university subscriptions). My suggestion was to avoid selecting for presentation papers with very similar content. Especially in the field of genome editing there has been a very rapid progress in the past few years resulting in a number of highly-cited articles which could appear very similar in content for a non-specialist. From the shortlist of 37 articles, students selected a topic they believed would be most interesting or easiest to explain. Presentations will be both written (in English, which is not the mother tongue of my students) and oral (in Slovenian, to establish and maintain Slovenian terminology in the field). <br /> <br /> ===List of articles for presentation===<br /> <br /> This is the list of top-cited papers from the broader field of Synthetic Biology that students chose for explanation in 2014/15 (sorted by year of publication):<br /> <br /> #[[A synthetic oscillatory network of transcriptional regulators]], Michael B. Elowitz &amp; Stanislas Leibler, Letters to Nature, 2000 - Valter Bergant<br /> #[[Construction of a genetic toggle switch in Escherichia coli]]. Gardner ''et al''., Nature, 2000 - Urban Bezeljak<br /> #[[Positive feedback in eukaryotic gene networks: cell differentiation by graded to binary response conversion]]. Becskei ''et al''., EMBO J, 2001 - Andreja Bratovš<br /> #[[Chemical synthesis of poliovirus cDNA: Generation of infectious virus in the absence of natural template]]. Jeronimo Cello ''et al''., Science,2002 - Veronika Jarc<br /> #[[Combinatorial synthesis of genetic networks]]. Guet C.C. ''et al'', Science, 2002 - Maja Remškar<br /> #[[Engineering a mevalonate pathway in Escherichia coli for production of terpenoids]] (2003) - Ana Kapraljević<br /> #Programmed population control by cell-cell communication and regulated killing. You et al, Nature (2004)[http://wiki.fkkt.uni-lj.si/index.php/7.Programmed_population_control_by_cell-cell_communication_and_regulated_killing] - Alja Zottel<br /> #Gene regulation at the single-cell level (2005) - Katarina Uršič<br /> #[[A synthetic multicellular system for programmed pattern formation]]. (2005) - Mitja Crček<br /> #[[Long-term monitoring of bacteria undergoing programmed population control in a microchemostat]]. Balagadde ''et al.'', ''Science'', 2005 - Jana Verbančič<br /> #[[Tuning genetic control through promoter engineering]], Hal Alper ''et al''., PNAS, 2005 - Špela Pohleven<br /> #[[Production of the antimalarial drug precursor artemisinic acid in engineered yeast ]]. Ro ''et al''., ''Nature''., 2006- Živa Marsetič<br /> #[[An improved zinc-finger nuclease architecture for highly specific genome editing]], Miller ''et al''., ''Nature Biotechnol''., 2007 - Eva Knapič<br /> #[[Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases]] (2008) - Tamara Marić<br /> #[[Synthetic protein scaffolds provide modular control over metabolic flux]]. Dueber ''et al''., Nature Biotechnology, 2009. - Ana Dolinar<br /> #[[Creation of a bacterial cell controlled by a chemically synthesized genome]]. Gibson, D. G. ''et al.'', Science, 2010 - Eva Lucija Kozak<br /> #[[A TALE nuclease architecture for efficient genome editing]], Miller ''et al'', ''Nature Biotechnol''., 2011 - Jernej Mustar<br /> #[[Multiplex genome engineering using CRISPR/Cas systems]] (2013) - Uroš Stupar<br /> #[[RNA-guided human genome engineering via Cas9]]. Mali ''et al''., Science, 2013 - Luka Smole<br /> #[[One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering (2013)]] - Andrej Vrankar<br /> <br /> <br /> ''Please link the title of each paper with your written seminar wiki page. Expand the citation according to the following example:<br /> ''<br /> #Emergent bistability by a growth-modulating positive feedback circuit. Tan et al., Nature Chem. Biol., 2009</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=ZFN_nukleaze&diff=8333 2013-10-15T18:00:37Z

<p>Uros.Stupar: /* UVOD */</p> <hr /> <div>=VISOKO USPEŠNA USMERJENA MUTAGENEZA S POMOČJO NUKLEAZ CINKOVEGA PRSTA V METULJU MONARHU=<br /> <br /> ==UVOD==<br /> <br /> Metulj monarh je žuželka, ki vsako jesen migrira na jug. Pomoga si s sončnim kompasom v povezavi z biološko uro, kar mu omogoča ohranjanje smeri na poti. Pri tem igrata pomembno vlogo dva različna CRY proteina. Mehanizem molekularne ure temelji na avtoregulatorni kontroli na nivoju transkripcije preko povratne zanke. Transkripcijska faktorja CLK in CYC sprožita transkripcijo per, tim in cry2 genov. To privede do nastanka proteinov, ki v citoplazmi tvorijo komplekse ter krožijo nazaj v jedro, kjer CRY2 inhibira transkripcijo.<br /> <br /> V naši študiji so se osredotočili na uvajanje mutacij v cry2 gen s pomočjo ''zinc-finger'' nukleaz (ZFN), ki ustvarjajo dedne poškodbe na DNA. ZFN so restrikcijske endonukleaze, ki lahko inducirajo mestno specifično prekinitev v obeh verigah DNA na želeni lokaciji v genomu. Prekinitve v obeh verigah DNA (DSB) se nato pri nizkih frakvencah popravljajo preko NHEJ poti, pri čemer pride do delecij in insercij v tarčnem mestu in posledično do spremembe bralnega okvirja. Induciranje DSB preko ZFN v kodirajočih zaporednjih proteinov, lahko povzroči nastanek nefunkcionalnih alelov.<br /> <br /> ==METODE IN REZULTATI==<br /> <br /> Najprej so z računalniškim algoritmom cry2 gen skenirali za tarčne sekvence, ki bi se skladale z arhivom modulov cinkovega prsta. Našli so visoko kvalitetno mesto v eksonu 2, ki vsebuje 2 prepoznavni regiji ob vmesnem mestu, ki vsebuje endogeno restrikcijsko mesto. Sprememba bralnega okvirja v tej regiji povzroči nefunkcionalnost proteina. Za vsak prepoznavni element so skonstruirali ZFN z restrikcijsko domeno FokI. Konstrukt je nastal tako, da so sekvence, ki kodirajo štiri prste ZFN klonirali v ekspresijski vektor pCS2.<br /> <br /> Celice DpN1 so transfecirali z različnimi dozami parov ZFN subkloniranih v ekspresijski vektor (pCS2) kompatibilen s celicami DpN1. Za določevanje uspešnosti transformacije so po amplifikaciji s PCR uporabili rezanje z restriktazo na mestu EagI, ki ga vsebuje divji tip gena. Tako dobimo pri divjem genu dva fragmenta. Po uvajanju mutacij preko mehanizma NHEJ restrikcijsko mesto EagI izgine in restriktaza ne more rezati, zato dobimo nerezan fragment. Učinkovitost ZFN za uvajanje mutacij je bila ocenjena na približno 12%. PCR fragmente, iz z ZNF transfeciranih celic, rezistentne na restrikcijo z EagI, so klonirali in sekvenirali.. Ugotovili so, da je v večini primerov prišlo do spremembe bralnega okvirja in posledično do skrajšanega proteina CRY2.<br /> <br /> Za generiranje CRY2 knockouta so uporabili tehniko, s katero so mutirali prekurzorje zarodne linije. To so storili z injiciranjem dveh mRNA s 5' kapo, ki kodirata obe ZFN v oplojena jajčeca v fazi ''enega jedra''. Injiciranje ZFN mRNA takoj po oploditvi pripomore k višji frekvenci mutacij v genomu, saj poteče mutacija zgodaj v razvoju osebka, v prekurzorskih celicah zarodne linije. <br /> <br /> Ta tehnika je omogočila visokofrekvenčno usmerjeno mutagenezo tako v somatskih kot tudi v zarodnih celicah. Prisotnost mutacij so testirali z restrikcijsko analizo tkiva, ki ne vpliva na preživetje ali plodnost osebkov ter sekvenirali mutirane alele. Ličinke z visoko frekvenco mutacij v somatskih celicah so gojili naprej do stadija odrasle živali, da bi lahko preučili ali se lahko te mutacije prenašajo na potomce. Odrasle osebke so križali z divjimi in analizirali potomce. Mutirane ličinke so odkrili le med potomci dveh moških staršev in sicer s frekvencama 39,2% in 50%. <br /> <br /> Za karakterizacijo narave ZFN-induciranih mutacij zarodne linije so klonirali in sekvenirali PCR fragmente, ki ustrezajo mutiranim alelom iz vsakega potomstva. V vsakem so našli po eno mutacijo. Ena mutacija je bila delecija 4 bp, druga pa insercija 2 bp, obe pa sta povzročili premik bralnega okvirja in posledično skrajšan protein CRY2, kar so potrdili tudi z izvedbo prenosa western. <br /> <br /> Kot je bilo pričakovati pri knockout linijah biološka ura ni delovala pravilno medtem ko pri divjem tipu in pri heterozigotih ni bilo težav. Da bi ugotovili CRY2 vpliv na biološko uro so s kvantitativnim PCR v realnem času kvantificirali ekspresijo per in tim genov v knockout linijah. Ekspresija obeh je bila močno povečana, kar je dokaz, da CRY2 in vivo deluje kot transkripcijski represor za gene, ki vplivajo na biološko uro.<br /> <br /> ==ZAKLJUČEK==<br /> <br /> Metoda usmerjene mutageneze z ZFN je zelo specifična in hkrati tudi zelo učinkovita, če jo izvedemo v pravem času. Injiciranje ZFN mRNA v fazi ''enega jedra'', v mesto kjer se v tej fazi nahajajo prekurzorske celice zarodne linije, je vzrok za visoko učinkovitost mutageneze tako v somatskih kot tudi v zarodnih celicah. Tako se mutacije z visoko frekvenco prenašajo tudi v naslednje generacije potomcev.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=ZFN_nukleaze&diff=8332 2013-10-15T18:00:00Z

<p>Uros.Stupar: /* METODE IN REZULTATI */</p> <hr /> <div>=VISOKO USPEŠNA USMERJENA MUTAGENEZA S POMOČJO NUKLEAZ CINKOVEGA PRSTA V METULJU MONARHU=<br /> <br /> ==UVOD==<br /> <br /> Metulj monarh je žuželka, ki vsako jesen migrira na jug. Pomoga si s sončnim kompasom v povezavi z biološko uro, kar mu omogoča ohranjanje smeri na poti. Pri tem igrata pomembno vlogo dva različna CRY proteina. Mehanizem molekularne ure temelji na avtoregulatorni kontroli na nivoju transkripcije preko povratne zanke. Transkripcijska faktorja CLK in CYC sprožita transkripcijo per, tim in cry2 genov. To privede do nastanka proteinov, ki v citoplazmi tvorijo komplekse ter krožijo nazaj v jedro, kjer CRY2 inhibira transkripcijo.<br /> V naši študiji so se osredotočili na uvajanje mutacij v cry2 gen s pomočjo ''zinc-finger'' nukleaz (ZFN), ki ustvarjajo dedne poškodbe na DNA. ZFN so restrikcijske endonukleaze, ki lahko inducirajo mestno specifično prekinitev v obeh verigah DNA na želeni lokaciji v genomu. Prekinitve v obeh verigah DNA (DSB) se nato pri nizkih frakvencah popravljajo preko NHEJ poti, pri čemer pride do delecij in insercij v tarčnem mestu in posledično do spremembe bralnega okvirja. Induciranje DSB preko ZFN v kodirajočih zaporednjih proteinov, lahko povzroči nastanek nefunkcionalnih alelov. <br /> <br /> ==METODE IN REZULTATI==<br /> <br /> Najprej so z računalniškim algoritmom cry2 gen skenirali za tarčne sekvence, ki bi se skladale z arhivom modulov cinkovega prsta. Našli so visoko kvalitetno mesto v eksonu 2, ki vsebuje 2 prepoznavni regiji ob vmesnem mestu, ki vsebuje endogeno restrikcijsko mesto. Sprememba bralnega okvirja v tej regiji povzroči nefunkcionalnost proteina. Za vsak prepoznavni element so skonstruirali ZFN z restrikcijsko domeno FokI. Konstrukt je nastal tako, da so sekvence, ki kodirajo štiri prste ZFN klonirali v ekspresijski vektor pCS2.<br /> <br /> Celice DpN1 so transfecirali z različnimi dozami parov ZFN subkloniranih v ekspresijski vektor (pCS2) kompatibilen s celicami DpN1. Za določevanje uspešnosti transformacije so po amplifikaciji s PCR uporabili rezanje z restriktazo na mestu EagI, ki ga vsebuje divji tip gena. Tako dobimo pri divjem genu dva fragmenta. Po uvajanju mutacij preko mehanizma NHEJ restrikcijsko mesto EagI izgine in restriktaza ne more rezati, zato dobimo nerezan fragment. Učinkovitost ZFN za uvajanje mutacij je bila ocenjena na približno 12%. PCR fragmente, iz z ZNF transfeciranih celic, rezistentne na restrikcijo z EagI, so klonirali in sekvenirali.. Ugotovili so, da je v večini primerov prišlo do spremembe bralnega okvirja in posledično do skrajšanega proteina CRY2.<br /> <br /> Za generiranje CRY2 knockouta so uporabili tehniko, s katero so mutirali prekurzorje zarodne linije. To so storili z injiciranjem dveh mRNA s 5' kapo, ki kodirata obe ZFN v oplojena jajčeca v fazi ''enega jedra''. Injiciranje ZFN mRNA takoj po oploditvi pripomore k višji frekvenci mutacij v genomu, saj poteče mutacija zgodaj v razvoju osebka, v prekurzorskih celicah zarodne linije. <br /> <br /> Ta tehnika je omogočila visokofrekvenčno usmerjeno mutagenezo tako v somatskih kot tudi v zarodnih celicah. Prisotnost mutacij so testirali z restrikcijsko analizo tkiva, ki ne vpliva na preživetje ali plodnost osebkov ter sekvenirali mutirane alele. Ličinke z visoko frekvenco mutacij v somatskih celicah so gojili naprej do stadija odrasle živali, da bi lahko preučili ali se lahko te mutacije prenašajo na potomce. Odrasle osebke so križali z divjimi in analizirali potomce. Mutirane ličinke so odkrili le med potomci dveh moških staršev in sicer s frekvencama 39,2% in 50%. <br /> <br /> Za karakterizacijo narave ZFN-induciranih mutacij zarodne linije so klonirali in sekvenirali PCR fragmente, ki ustrezajo mutiranim alelom iz vsakega potomstva. V vsakem so našli po eno mutacijo. Ena mutacija je bila delecija 4 bp, druga pa insercija 2 bp, obe pa sta povzročili premik bralnega okvirja in posledično skrajšan protein CRY2, kar so potrdili tudi z izvedbo prenosa western. <br /> <br /> Kot je bilo pričakovati pri knockout linijah biološka ura ni delovala pravilno medtem ko pri divjem tipu in pri heterozigotih ni bilo težav. Da bi ugotovili CRY2 vpliv na biološko uro so s kvantitativnim PCR v realnem času kvantificirali ekspresijo per in tim genov v knockout linijah. Ekspresija obeh je bila močno povečana, kar je dokaz, da CRY2 in vivo deluje kot transkripcijski represor za gene, ki vplivajo na biološko uro.<br /> <br /> ==ZAKLJUČEK==<br /> <br /> Metoda usmerjene mutageneze z ZFN je zelo specifična in hkrati tudi zelo učinkovita, če jo izvedemo v pravem času. Injiciranje ZFN mRNA v fazi ''enega jedra'', v mesto kjer se v tej fazi nahajajo prekurzorske celice zarodne linije, je vzrok za visoko učinkovitost mutageneze tako v somatskih kot tudi v zarodnih celicah. Tako se mutacije z visoko frekvenco prenašajo tudi v naslednje generacije potomcev.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=ZFN_nukleaze&diff=8331 2013-10-15T17:58:05Z

<p>Uros.Stupar: </p> <hr /> <div>=VISOKO USPEŠNA USMERJENA MUTAGENEZA S POMOČJO NUKLEAZ CINKOVEGA PRSTA V METULJU MONARHU=<br /> <br /> ==UVOD==<br /> <br /> Metulj monarh je žuželka, ki vsako jesen migrira na jug. Pomoga si s sončnim kompasom v povezavi z biološko uro, kar mu omogoča ohranjanje smeri na poti. Pri tem igrata pomembno vlogo dva različna CRY proteina. Mehanizem molekularne ure temelji na avtoregulatorni kontroli na nivoju transkripcije preko povratne zanke. Transkripcijska faktorja CLK in CYC sprožita transkripcijo per, tim in cry2 genov. To privede do nastanka proteinov, ki v citoplazmi tvorijo komplekse ter krožijo nazaj v jedro, kjer CRY2 inhibira transkripcijo.<br /> V naši študiji so se osredotočili na uvajanje mutacij v cry2 gen s pomočjo ''zinc-finger'' nukleaz (ZFN), ki ustvarjajo dedne poškodbe na DNA. ZFN so restrikcijske endonukleaze, ki lahko inducirajo mestno specifično prekinitev v obeh verigah DNA na želeni lokaciji v genomu. Prekinitve v obeh verigah DNA (DSB) se nato pri nizkih frakvencah popravljajo preko NHEJ poti, pri čemer pride do delecij in insercij v tarčnem mestu in posledično do spremembe bralnega okvirja. Induciranje DSB preko ZFN v kodirajočih zaporednjih proteinov, lahko povzroči nastanek nefunkcionalnih alelov. <br /> <br /> ==METODE IN REZULTATI==<br /> <br /> Najprej so z računalniškim algoritmom cry2 gen skenirali za tarčne sekvence, ki bi se skladale z arhivom modulov cinkovega prsta. Našli so visoko kvalitetno mesto v eksonu 2, ki vsebuje 2 prepoznavni regiji ob vmesnem mestu, ki vsebuje endogeno restrikcijsko mesto. Sprememba bralnega okvirja v tej regiji povzroči nefunkcionalnost proteina. Za vsak prepoznavni element so skonstruirali ZFN z restrikcijsko domeno FokI. Konstrukt je nastal tako, da so sekvence, ki kodirajo štiri prste ZFN klonirali v ekspresijski vektor pCS2.<br /> Celice DpN1 so transfecirali z različnimi dozami parov ZFN subkloniranih v ekspresijski vektor (pCS2) kompatibilen s celicami DpN1. Za določevanje uspešnosti transformacije so po amplifikaciji s PCR uporabili rezanje z restriktazo na mestu EagI, ki ga vsebuje divji tip gena. Tako dobimo pri divjem genu dva fragmenta. Po uvajanju mutacij preko mehanizma NHEJ restrikcijsko mesto EagI izgine in restriktaza ne more rezati, zato dobimo nerezan fragment. Učinkovitost ZFN za uvajanje mutacij je bila ocenjena na približno 12%. PCR fragmente, iz z ZNF transfeciranih celic, rezistentne na restrikcijo z EagI, so klonirali in sekvenirali.. Ugotovili so, da je v večini primerov prišlo do spremembe bralnega okvirja in posledično do skrajšanega proteina CRY2.<br /> Za generiranje CRY2 knockouta so uporabili tehniko, s katero so mutirali prekurzorje zarodne linije. To so storili z injiciranjem dveh mRNA s 5' kapo, ki kodirata obe ZFN v oplojena jajčeca v fazi ''enega jedra''. Injiciranje ZFN mRNA takoj po oploditvi pripomore k višji frekvenci mutacij v genomu, saj poteče mutacija zgodaj v razvoju osebka, v prekurzorskih celicah zarodne linije. <br /> Ta tehnika je omogočila visokofrekvenčno usmerjeno mutagenezo tako v somatskih kot tudi v zarodnih celicah. Prisotnost mutacij so testirali z restrikcijsko analizo tkiva, ki ne vpliva na preživetje ali plodnost osebkov ter sekvenirali mutirane alele. Ličinke z visoko frekvenco mutacij v somatskih celicah so gojili naprej do stadija odrasle živali, da bi lahko preučili ali se lahko te mutacije prenašajo na potomce. Odrasle osebke so križali z divjimi in analizirali potomce. Mutirane ličinke so odkrili le med potomci dveh moških staršev in sicer s frekvencama 39,2% in 50%. <br /> Za karakterizacijo narave ZFN-induciranih mutacij zarodne linije so klonirali in sekvenirali PCR fragmente, ki ustrezajo mutiranim alelom iz vsakega potomstva. V vsakem so našli po eno mutacijo. Ena mutacija je bila delecija 4 bp, druga pa insercija 2 bp, obe pa sta povzročili premik bralnega okvirja in posledično skrajšan protein CRY2, kar so potrdili tudi z izvedbo prenosa western. <br /> Kot je bilo pričakovati pri knockout linijah biološka ura ni delovala pravilno medtem ko pri divjem tipu in pri heterozigotih ni bilo težav. Da bi ugotovili CRY2 vpliv na biološko uro so s kvantitativnim PCR v realnem času kvantificirali ekspresijo per in tim genov v knockout linijah. Ekspresija obeh je bila močno povečana, kar je dokaz, da CRY2 in vivo deluje kot transkripcijski represor za gene, ki vplivajo na biološko uro.<br /> <br /> ==ZAKLJUČEK==<br /> <br /> Metoda usmerjene mutageneze z ZFN je zelo specifična in hkrati tudi zelo učinkovita, če jo izvedemo v pravem času. Injiciranje ZFN mRNA v fazi ''enega jedra'', v mesto kjer se v tej fazi nahajajo prekurzorske celice zarodne linije, je vzrok za visoko učinkovitost mutageneze tako v somatskih kot tudi v zarodnih celicah. Tako se mutacije z visoko frekvenco prenašajo tudi v naslednje generacije potomcev.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=ZFN_nukleaze&diff=8330 2013-10-15T17:57:35Z

<p>Uros.Stupar: New page: =VISOKO USPEŠNA USMERJENA MUTAGENEZA S POMOČJO NUKLEAZ CINKOVEGA PRSTA V METULJU MONARHU= ==UVOD== Metulj monarh je žuželka, ki vsako jesen migrira na jug. Pomoga si s sončnim kompa...</p> <hr /> <div>=VISOKO USPEŠNA USMERJENA MUTAGENEZA S POMOČJO NUKLEAZ CINKOVEGA PRSTA V METULJU MONARHU=<br /> <br /> ==UVOD==<br /> <br /> Metulj monarh je žuželka, ki vsako jesen migrira na jug. Pomoga si s sončnim kompasom v povezavi z biološko uro, kar mu omogoča ohranjanje smeri na poti. Pri tem igrata pomembno vlogo dva različna CRY proteina. Mehanizem molekularne ure temelji na avtoregulatorni kontroli na nivoju transkripcije preko povratne zanke. Transkripcijska faktorja CLK in CYC sprožita transkripcijo per, tim in cry2 genov. To privede do nastanka proteinov, ki v citoplazmi tvorijo komplekse ter krožijo nazaj v jedro, kjer CRY2 inhibira transkripcijo.<br /> V naši študiji so se osredotočili na uvajanje mutacij v cry2 gen s pomočjo ''zinc-finger'' nukleaz (ZFN), ki ustvarjajo dedne poškodbe na DNA. ZFN so restrikcijske endonukleaze, ki lahko inducirajo mestno specifično prekinitev v obeh verigah DNA na želeni lokaciji v genomu. Prekinitve v obeh verigah DNA (DSB) se nato pri nizkih frakvencah popravljajo preko NHEJ poti, pri čemer pride do delecij in insercij v tarčnem mestu in posledično do spremembe bralnega okvirja. Induciranje DSB preko ZFN v kodirajočih zaporednjih proteinov, lahko povzroči nastanek nefunkcionalnih alelov. <br /> METODE IN REZULTATI<br /> Najprej so z računalniškim algoritmom cry2 gen skenirali za tarčne sekvence, ki bi se skladale z arhivom modulov cinkovega prsta. Našli so visoko kvalitetno mesto v eksonu 2, ki vsebuje 2 prepoznavni regiji ob vmesnem mestu, ki vsebuje endogeno restrikcijsko mesto. Sprememba bralnega okvirja v tej regiji povzroči nefunkcionalnost proteina. Za vsak prepoznavni element so skonstruirali ZFN z restrikcijsko domeno FokI. Konstrukt je nastal tako, da so sekvence, ki kodirajo štiri prste ZFN klonirali v ekspresijski vektor pCS2.<br /> Celice DpN1 so transfecirali z različnimi dozami parov ZFN subkloniranih v ekspresijski vektor (pCS2) kompatibilen s celicami DpN1. Za določevanje uspešnosti transformacije so po amplifikaciji s PCR uporabili rezanje z restriktazo na mestu EagI, ki ga vsebuje divji tip gena. Tako dobimo pri divjem genu dva fragmenta. Po uvajanju mutacij preko mehanizma NHEJ restrikcijsko mesto EagI izgine in restriktaza ne more rezati, zato dobimo nerezan fragment. Učinkovitost ZFN za uvajanje mutacij je bila ocenjena na približno 12%. PCR fragmente, iz z ZNF transfeciranih celic, rezistentne na restrikcijo z EagI, so klonirali in sekvenirali.. Ugotovili so, da je v večini primerov prišlo do spremembe bralnega okvirja in posledično do skrajšanega proteina CRY2.<br /> Za generiranje CRY2 knockouta so uporabili tehniko, s katero so mutirali prekurzorje zarodne linije. To so storili z injiciranjem dveh mRNA s 5' kapo, ki kodirata obe ZFN v oplojena jajčeca v fazi ''enega jedra''. Injiciranje ZFN mRNA takoj po oploditvi pripomore k višji frekvenci mutacij v genomu, saj poteče mutacija zgodaj v razvoju osebka, v prekurzorskih celicah zarodne linije. <br /> Ta tehnika je omogočila visokofrekvenčno usmerjeno mutagenezo tako v somatskih kot tudi v zarodnih celicah. Prisotnost mutacij so testirali z restrikcijsko analizo tkiva, ki ne vpliva na preživetje ali plodnost osebkov ter sekvenirali mutirane alele. Ličinke z visoko frekvenco mutacij v somatskih celicah so gojili naprej do stadija odrasle živali, da bi lahko preučili ali se lahko te mutacije prenašajo na potomce. Odrasle osebke so križali z divjimi in analizirali potomce. Mutirane ličinke so odkrili le med potomci dveh moških staršev in sicer s frekvencama 39,2% in 50%. <br /> Za karakterizacijo narave ZFN-induciranih mutacij zarodne linije so klonirali in sekvenirali PCR fragmente, ki ustrezajo mutiranim alelom iz vsakega potomstva. V vsakem so našli po eno mutacijo. Ena mutacija je bila delecija 4 bp, druga pa insercija 2 bp, obe pa sta povzročili premik bralnega okvirja in posledično skrajšan protein CRY2, kar so potrdili tudi z izvedbo prenosa western. <br /> Kot je bilo pričakovati pri knockout linijah biološka ura ni delovala pravilno medtem ko pri divjem tipu in pri heterozigotih ni bilo težav. Da bi ugotovili CRY2 vpliv na biološko uro so s kvantitativnim PCR v realnem času kvantificirali ekspresijo per in tim genov v knockout linijah. Ekspresija obeh je bila močno povečana, kar je dokaz, da CRY2 in vivo deluje kot transkripcijski represor za gene, ki vplivajo na biološko uro.<br /> <br /> ==ZAKLJUČEK==<br /> <br /> Metoda usmerjene mutageneze z ZFN je zelo specifična in hkrati tudi zelo učinkovita, če jo izvedemo v pravem času. Injiciranje ZFN mRNA v fazi ''enega jedra'', v mesto kjer se v tej fazi nahajajo prekurzorske celice zarodne linije, je vzrok za visoko učinkovitost mutageneze tako v somatskih kot tudi v zarodnih celicah. Tako se mutacije z visoko frekvenco prenašajo tudi v naslednje generacije potomcev.</div>

Uros.Stupar https://wiki.fkkt.uni-lj.si/index.php?title=Seminarji_TehDNA&diff=8329 2013-10-15T17:55:54Z

<p>Uros.Stupar: </p> <hr /> <div>Seminarje iz Tehnologije DNA bo v študijskem letu 2013/14 vodila asist. dr. Helena Čelešnik.<br /> <br /> Seznam tem za seminarje:<br /> <br /> # Mutageneza (16.10.) (povzetek in porocilo lahko oddate v pon., 14.10), 3 seminarji: 1. Urban Bezeljak (CRISPR/Cas9) 2. Uroš Stupar (ZFN nukleaze) 3. Helena Vajović (tarčna mutageneza)<br /> # Izražanje na površini (23.10.), 3 seminarji: 1. Mitja Crček (Ribosome display), 2. Klara Tereza Novoselc (Phage display) 3. Živa Marsetič<br /> # Dvohibridni sistemi (30.10.), 3 seminarji: 1. Katja Kovačič 2. Barbara Žužek 3. Bernarda Majc<br /> # Mutageneza, izražanje na površini ali dvohibridni sistemi (6.11.), 3 seminarji: 1. Valter Bergant (scFv phage display), 2. Ana Kapraljević, 3. Tjaša Blatnik<br /> # GSO v agronomiji (13.11.), 3 seminarji: 1. Niki Bursič, 2. Petra Malavašič, 3. Jernej Mustar<br /> # Transgenske živali (27.11.), 3 seminarji: 1. Andrea Grof, 2. Eva Lucija Kozak, 3. Špela Pohleven<br /> # Izvorne celice (4.12.), 4 seminarji: 1. Sara Primec, 2. Alja Zottel, 3. Tjaša Goričan, 4. Rok Štemberger<br /> # DNA-diagnostika (11.12.), 4 seminarji: 1. Tina Gregorič , 2. Eva Knapič, 3. Veronika Jarc, 4. Jana Verbančič<br /> # Forenzika, arheologija, sistematika (18.12.), 3 seminarji: 1. Matja Zalar, 2. Andreja Bratovš, 3. Maja Remškar<br /> # Mikromreže, genomike (8.1.), 3 seminarji: 1. Andrej Vrankar, 2. Filip Kolenc 3. Nastja Štemberger<br /> # Gensko zdravljenje s. lat. (15.1.), 3 seminarji: 1. Ana Dolinar 2. Staša Komljenovič, 3. Katarina Uršič<br /> <br /> <br /> '''IZBIRANJE ČLANKOV ZA SEMINARJE:<br /> Ni nujno, da je metoda, ki jo želimo predstaviti, sama tematika izbranega članka. Zaželeno je, da članek obravnava neko biološko temo, pri raziskovanju le-te pa avtorji uporabljajo metodo, ki jo želimo predstaviti.''' <br /> <br /> <br /> '''POVZETKI ZA SEMINARJE 14.10.2013'''<br /> <br /> 1. Urban Bezeljak ([[CRISPR/Cas9]])<br /> <br /> 2. Uroš Stupar ([[ZFN nukleaze]])<br /> <br /> 3. Helena Vajović ([[tarčna mutageneza]])</div>

Uros.Stupar