Wiki FKKT - User contributions [en]

2015-bionano-seminar

2015-06-07T14:03:48Z

Luka Smole:

= Bionanotehnologija- seminar =
doc. dr. Gregor Gunčar, K2.022

== Seznam seminarjev ==
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Avtor 1'''
| align="center" style="background:#f0f0f0;"|'''Avtor 2'''
| align="center" style="background:#f0f0f0;"|'''Naslov seminarja'''
| align="center" style="background:#f0f0f0;"|'''Datum za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 2'''
|-
| Anže Prašnikar||Monika Praznik ||||29.03.||31.03.||Aneja Tuljak||Angelika Vižintin
|-
| Varja Božič||Eva Knapič||Razgradljivi kondomi s protimikrobno zaščito||29.03.||31.03.||Eva Udovič||Maja Grdadolnik
|-
| Belkisa Velagić||Aleksander Benčič||Avtomobilski encimski katalizator||29.03.||31.03.||Nika Kurinčič||Tjaša Goričan
|-
| Naja Vrankar||Valter Bergant||Modifikacija črevesne mikroflore pri debelosti||31.03.||02.04.||Nataša Žigante||Luka Smole
|-
| Tilen Volčanšek||Veronika Jarc||||31.03.||02.04.||Anže Prašnikar||Jakob Gašper Lavrenčič
|-
| Tanja Lipec||Iza Ogris||Test občutljivosti na gluten z neprebavljivo kapsulo||31.03.||02.04.||Varja Božič||Klara Tereza Novoselc
|-
| Katja Lovrin||Mitja Crček||Uporaba nitrifikacijskih encimov pri kmetovanju||05.04.||07.04.||Belkisa Velagić||Monika Praznik
|-
| Saša Balažic||Urban Javoršek ||||05.04.||07.04.||Naja Vrankar||Eva Knapič
|-
| Urban Borštnik||Sara Primec||||05.04.||07.04.||Tilen Volčanšek||Aleksander Benčič
|-
| Nives Ahlin||Kim Kos||||12.04.||14.04.||Tanja Lipec||Valter Bergant
|-
| Matic Bevec||Estera Merljak||Antimaček®||12.04.||14.04.||Katja Lovrin||Veronika Jarc
|-
| Vida Špindler||Jernej Pušnik||||12.04.||14.04.||Saša Balažic||Iza Ogris
|-
| Jasmina Sedmak||Maxi Sagmeister||Brezžični zobni nanobiosenzor ||19.04.||21.04.||Urban Borštnik||Mitja Crček
|-
| Sanja Popović||Benjamin Bajželj||||19.04.||21.04.||Nives Ahlin||Urban Javoršek
|-
| Blaž Komar||Alja Zottel||||19.04.||21.04.||Matic Bevec||Sara Primec
|-
| Blaž Perič||Katarina Uršič||Biorazgradljivi žvečilni gumi z antibakterijskimi lastnostmi||03.05.||05.05.||Simon Preložnik||Kim Kos
|-
| Simon Preložnik||Maja Remškar||Preprost dostavni sistem za omega-3 maščobne kisline||03.05.||05.05.||Jasmina Sedmak||Estera Merljak
|-
| Aneja Tuljak||Tina Gregorič||Stekleničke z biosenzorjem za detekcijo ''E.coli''||03.05.||05.05.||Sanja Popović||Jernej Pušnik
|-
| Damir Hamulić||Anita Kustec||Pretvorba CO2 v uporabne proizvode s pomočjo umetne fotosinteze||10.05.||12.05.||Blaž Komar||Maxi Sagmeister
|-
| Janja Fortin||Tina Snoj|| Zaviralec nadležnih posledic komarjevega pika (Antikomarin) ||10.05.||12.05.||Blaž Perič||Benjamin Bajželj
|-
| Rajko Vnuk||Mojca Banič||||10.05.||12.05.||Vida Špindler||Alja Zottel
|-
| Rok Grm||Ajda Rojc||||17.05.||19.05.||Kaja Javoršek||Katarina Uršič
|-
| Kristina Gavranić||Barbara Žužek||Protimikrobni žvečilni gumi s hidroksiapatitnimi nanodelci za remineralizacijo zobne sklenine||17.05.||19.05.||Damir Hamulić||Maja Remškar
|-
| Urška Mohorič||Griša Prinčič||Verižna reakcija s polimerazo na osnovi denaturacije z magnetnimi nanodelci||17.05.||19.05.||Janja Fortin||Tina Gregorič
|-
| Maja Ramić||Nejc Petrišič||||24.05.||26.05.||Rajko Vnuk||Anita Kustec
|-
| Barbara Jeras||Tamara Marić||||24.05.||26.05.||Rok Grm||Tina Snoj
|-
| Matic Urlep||Samo Zakotnik||Uporaba optogenetike za lajšanje simptomov Parkinsonove bolezni||24.05.||26.05.||Kristina Gavranić||Mojca Banič
|-
| Urban Verbič||Angelika Vižintin||||31.05.||02.06.||Urška Mohorič||Ajda Rojc
|-
| Nataša Žigante||Maja Grdadolnik||||31.05.||02.06.||Maja Ramić||Barbara Žužek
|-
| Kaja Javoršek||Tjaša Goričan||Kontaktne leče, ki preventivno ščitijo pred očesnimi boleznimi (proti slepoti)||31.05.||02.06.||Barbara Jeras||Griša Prinčič
|-
| Eva Udovič||Luka Smole||Celični vsadek za nadzor ravni glukoze v krvi||07.06.||09.06.||Matic Urlep||Nejc Petrišič
|-
| Nika Kurinčič||Jakob Gašper Lavrenčič||||07.06.||09.06.||Urban Verbič||Tamara Marić
|-
| Klara Tereza Novoselc||||||07.06.||09.06.||Samo Zakotnik||
|}

== Gradivo za predavanja ==
Gradivo za predavanja najdete v [http://ucilnica.fkkt.uni-lj.si/ spletni učilnici].

==Naloga==
'''Vaša naloga je: '''
Po dva študenta skupaj pripravita projektno nalogo iz področja Bionanotehnologije. Najpomembnejša je originalna ideja za nek izvedljiv projekt.
Predlagana struktura:
* Uvod
* Predstavitev problema, znanstvena izhodišča, cilji
* Izvedba projekta, metodologija, tehnike, materiali, vprašanja, hipoteze
* Literatura

Za pripravo seminarja velja naslednje: 
* Prva stran seminarja naj vsebuje naslov projekta, avtorje, povzetek (od 130 do 160 besed) in grafični povzetek (čez približno pol strani)
* Seminar pripravite v obliki seminarske naloge na ~5 straneh A4 (pisava 12, enojni razmak, 2,5 cm robovi). Zelo pomembno je, da je obseg od 1500 do 2000 besed . Seminarska naloga mora vsebovati najmanj tri slike. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko. 
* Seminar oddajte do datuma oddaje, ki je naveden v tabeli v elektronski obliki z uporabo [http://bio.ijs.si/~zajec/poslji/ tega obrazca].
* Vsi seminarji so v elektronski obliki dostopni [http://bio.ijs.si/~zajec/poslji/bioseminar/ tukaj].
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 20 minut, predstavitev pa ne sme biti krajša od 15 minut (popust :-)). Nalogo predstavita oba študenta (razdelita si čas). Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava. Recenzenti podajo pripombe k projektu in postavijo po dve vprašanji.
* Na dan predstavitve morate docentu še pred predstavitvijo oddati končno verzijo seminarja v enem izvodu, elektronsko verzijo seminarja in predstavitev pa oddati na strežnik na dan predstavitve do polnoči.

==Imena datotek==
Prosim vas, da vse datoteke poimenujete po naslednjem receptu:
* 19_nano_Priimek1_Priimek2.doc(x) za seminar, npr. 19_nano_Craik_Venter.docx
* 19_nano_Priimek1_Priimek2.ppt(x) za prezentacijo, npr. 19_nano_Craik_Venter.pptx

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [https://docs.google.com/forms/d/1WdCXoXo1zkRrVlLKIcEV1z_MyhavU-3ERBm9n2oiawI/viewform recenzentsko poročilo] na spletu. Recenzentsko poročilo morate oddati najkasneje do predstavitve seminarja.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://docs.google.com/forms/d/1ToLPn78T9W3G6Hm5hV0mLseFYghiLQMlRPGb0J5zft8/viewform mnenje] najkasneje v sedmih dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.
Na [http://bit.ly/bntmnenja tej strani] lahko preverite, če ste svoje mnenje za določen seminar že oddali.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se držite ene same. V seminarskih nalogah in diplomskih nalogah FKKT uprabljajte shemo citiranja, ki je pobarvana zeleno.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.zveza-zotks.si/gzm/dokumenti/literatura.html Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue, C., Glass, J. I., Alperovich, N., Pieper, R., Parmar, P. P., Hutchison III, C. A., Smith, H. O. in Venter, J. C.
Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne). Navesti morate tudi vse avtorje dela, razen v primeru, ko jih je 10 ali več. Takrat navedite le prvih devet, za ostale pa uporabite okrajšavo in sod. (in sodelavci). Pred zadnjim avtorjem naj bo vedno besedica "in".

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

Z EEG nadzorovano izražanje transgena preko brezžično napajanega optogenetskega celičnega vsadka.

2015-06-04T15:08:28Z

Luka Smole:

==UVOD==
Optogenetika je tehnologija, ki omogoča hiter in natančen tarčni nadzor nad želenimi dogodki v bioloških sistemih na molekularni ravni. Gre za kombinacijo genetskih in optičnih metod, ki povzročijo ali prekinejo delovanje točno določenih procesov v celicah. V najširšem pomenu optogenetika predstavlja orodje za tarčno regulacijo, ki je odvisna od svetlobe in posledično izvaja neko efektorsko funkcijo.
Raziskavo, ki je povzeta v seminarju so opravili raziskovalci iz raziskovalne skupine Martina Fusseneggerja z ETH v Zurichu, ki je svetovno znan znanstvenik s področja sintezne biologije in molekularne biotehnologije.

==RAZISKAVA==
Cilj raziskave je bil načrtovanje svetlobno občutljivega genetskega vezja v sesalskih celicah za vstavitev v kamrice z LED diodami, ki inducirajo izražanje gena za sekretorno alkalno fosfatazo (SEAP). Kamrice služijo kot podkožni vsadek v miškah, v katerih z uporabo posebnega sprejemniškega vezja preko EEG z različnimi aktivnostmi možganskih valov sprožijo delovanje LED diod in posledično izražanje SEAP iz načrtovanih celic.

===Načrtovanje svetlobno občutljivega genetskega vezja===
Raziskovalci so izdelali posebne kamrice z LED lučkami, ki so svetile s svetlobo v NIR spektru valovne dolžine 700 nm. V kamricah so bile nacepljene sesalske celice HEK-293F, ki so vsebovale načrtovano genetsko vezje. Celice HEK-293F se od HEK-293T razlikujejo po tem, da lahko rastejo v suspenzijski kulturi brez dodanega seruma. To dejstvo je pomembno za gojenje teh celic v načrtovanih kamricah. Gojitvene kamrice s celicami so bile od zunanjega okolja ločene s polprepustno membrano, ki ima prepustnost za molekule, manjše od 300 kDa.
Raziskovalci so načrtovali vnos signalizacijske poti v sesalske celice, ki temelji na izražanju sekundarnega obveščevalca c-di-GMP. Za produkcijo c-di-GMP so uporabili skrajšano različico svetlobno občutljivega večdomenskega proteina BphG1 iz bakterije Rhodobacter sphaeroides brez fosfodiesterazne aktivnosti. Ta protein vsebuje N-terminalno na NIR svetlobo občutljivo domeno in C-terminalno digvanilat ciklazno domeno (DGC). Protein BphG1 ima tudi fosfodiesterazno aktivnost (PDE), ki omogoča uravnavanje koncentracije c-di-GMP, ki ima v bakterijskih celicah vlogo uravnavanja prehoda celic iz premikajočega stanja v tvorbo biofilma. Skrajšano različico BphG1, ki katalizira nastanek sekundarnega obveščevalca c-di-GMP iz GTP so poimenovali DGCL . Preko tega obveščevalca pride do aktivacije proteina STING na endoplazemskem retikulumu (ang. »stimulator of interferon genes«), ki povzroči fosforilacijo IRF3 (ang. »interferon-regulatory factor 3«) preko TBK1 (ang. »tank-binding kinase 1«). Nato pride do premestitve fosforiliranega IRF3 v jedro, kjer z vezavo na specifične operatorje aktivira promotorje interferona I. Pod kontrolo promotorja interferona I je bilo izražanje SEAP (ang. »Secreted embryonic alkaline phosphatase«), ki je pogosto uporabljan poročevalski sistem. Temelji na izražanju alkalne fosfataze, ki se izloča v gojišče, ob dodatku substrata pa lahko zaznavamo produkt.

===Z EEG nadzorovan elektro-optogenetski sistem===
Z mislimi sprožene elektrofiziološke signale so preko EEG pošiljali v BCI (ang. »brain-computer interface«), ki je bil naravnan tako, da ob prehodu EEG signala nad določeno vrednost brezžižčno sproži oddajanje svetlobe v vsadku z lučkami NIR.
Sistem za nadzor nad elektro-optogenetskim sistemom temelji na različnih signalih EEG možganskih valov (surov signal v µV), ki ga človek sproži z različnimi stanji zavesti. Za učinkovit nadzor nad sistemom so identificirali za različna psihološka stanja specifične elektronske signale, ki služijo kot stikalo za sprožitev delovanja optogenetskega vezja. Osredotočili so se na stanje meditacije, ki so jo dosegli s priučenim sproščanjem. Drugo stanje je bila osredotočenost, ki so jo dosegli z igranjem računalniških iger. Ko je signal presegel določeno mejo je preko BCI prišlo do osvetlitve celic s pulzom NIR svetlobe. Tako so dosegli različna izražanja SEAP v celicah z načrtovanim optogenetskim vezjem.
Kot omenjeno zgoraj, BCI deluje kot generator polja, ki v odvisnosti od EEG signalov brezžično aktivira proizvajanje pulzov NIR svetlobe v podkožnem vsadku.

Brezžično napajani optogenetski vsadki predstavljajo modularen stik elektronike z živimi celicami, ki omogoča neposreden nadzor nad izražanjem genov preko električnih naprav. Pristop, ki je opisan v seminarju pa je stik možganske aktivnosti z elektrogenetsko napravo. Predstavlja nove možnosti načrtovanja elektromehanskih vsadkov, kot so srčni spodbujevalniki, očesne proteze in inzulinske mikročrpalke.

Z EEG nadzorovano izražanje transgena preko brezžično napajanega optogenetskega celičnega vsadka.

2015-06-04T15:03:44Z

Luka Smole: New page: ==UVOD== Optogenetika je tehnologija, ki omogoča hiter in natančen tarčni nadzor nad želenimi dogodki v bioloških sistemih na molekularni ravni. Gre za kombinacijo genetskih in optič...

==UVOD==
Optogenetika je tehnologija, ki omogoča hiter in natančen tarčni nadzor nad želenimi dogodki v bioloških sistemih na molekularni ravni. Gre za kombinacijo genetskih in optičnih metod, ki povzročijo ali prekinejo delovanje točno določenih procesov v celicah. V najširšem pomenu optogenetika predstavlja orodje za tarčno regulacijo, ki je odvisna od svetlobe in posledično izvaja neko efektorsko funkcijo.
Raziskavo, ki je povzeta v seminarju so opravili raziskovalci iz raziskovalne skupine Martina Fusseneggerja z ETH v Zurichu, ki je svetovno znan znanstvenik s področja sintezne biologije in molekularne biotehnologije.

==RAZISKAVA==
Cilj raziskave je bil načrtovanje svetlobno občutljivega genetskega vezja v sesalskih celicah za vstavitev v kamrice z LED diodami, ki inducirajo izražanje gena za sekretorno alkalno fosfatazo (SEAP). Kamrice služijo kot podkožni vsadek v miškah, v katerih z uporabo posebnega sprejemniškega vezja preko EEG z različnimi aktivnostmi možganskih valov sprožijo delovanje LED diod in posledično izražanje SEAP.
Načrtovanje svetlobno občutljivega genetskega vezja
Raziskovalci so izdelali posebne kamrice z LED lučkami, ki so svetile s svetlobo v NIR spektru valovne dolžine 700 nm. V kamricah so bile nacepljene sesalske celice HEK-293F, ki so vsebovale načrtovano genetsko vezje. Celice HEK-293F se od HEK-293T razlikujejo po tem, da lahko rastejo v suspenzijski kulturi brez dodanega seruma. To dejstvo je pomembno za gojenje teh celic v načrtovanih kamricah. Gojitvene kamrice s celicami so bile od zunanjega okolja ločene s polprepustno membrano, ki ima prepustnost za molekule, manjše od 300 kDa.
Raziskovalci so načrtovali vnos signalizacijske poti v sesalske celice, ki temelji na izražanju sekundarnega obveščevalca c-di-GMP. Za produkcijo c-di-GMP so uporabili skrajšano različico svetlobno občutljivega večdomenskega proteina BphG1 iz bakterije Rhodobacter sphaeroides brez fosfodiesterazne aktivnosti. Ta protein vsebuje N-terminalno na NIR svetlobo občutljivo domeno in C-terminalno digvanilat ciklazno domeno (DGC). Protein BphG1 ima tudi fosfodiesterazno aktivnost (PDE), ki omogoča uravnavanje koncentracije c-di-GMP, ki ima v bakterijskih celicah vlogo uravnavanja prehoda celic iz premikajočega stanja v tvorbo biofilma. Skrajšano različico BphG1, ki katalizira nastanek sekundarnega obveščevalca c-di-GMP iz GTP so poimenovali DGCL . Preko tega obveščevalca pride do aktivacije proteina STING na endoplazemskem retikulumu (ang. »stimulator of interferon genes«), ki povzroči fosforilacijo IRF3 (ang. »interferon-regulatory factor 3«) preko TBK1 (ang. »tank-binding kinase 1«). Nato pride do premestitve fosforiliranega IRF3 v jedro, kjer z vezavo na specifične operatorje aktivira promotorje interferona I. Pod kontrolo promotorja interferona I je bilo izražanje SEAP (ang. »Secreted embryonic alkaline phosphatase«), ki je pogosto uporabljan poročevalski sistem. Temelji na izražanju alkalne fosfataze, ki se izloča v gojišče, ob dodatku substrata pa lahko zaznavamo produkt.

===Z EEG nadzorovan elektro-optogenetski sistem===
Z mislimi sprožene elektrofiziološke signale so preko EEG pošiljali v BCI (ang. »brain-computer interface«), ki je bil naravnan tako, da ob prehodu EEG signala nad določeno vrednost brezžižčno sproži oddajanje svetlobe v vsadku z lučkami NIR.
Sistem za nadzor nad elektro-optogenetskim sistemom temelji na različnih signalih EEG možganskih valov (surov signal v µV), ki ga človek sproži z različnimi stanji zavesti. Za učinkovit nadzor nad sistemom so identificirali za različna psihološka stanja specifične elektronske signale, ki služijo kot stikalo za sprožitev delovanja optogenetskega vezja. Osredotočili so se na stanje meditacije, ki so jo dosegli s priučenim sproščanjem. Drugo stanje je bila osredotočenost, ki so jo dosegli z igranjem računalniških iger. Ko je signal presegel določeno mejo je preko BCI prišlo do osvetlitve celic s pulzom NIR svetlobe. Tako so dosegli različna izražanja SEAP v celicah z načrtovanim optogenetskim vezjem.
Kot omenjeno zgoraj, BCI deluje kot generator polja, ki v odvisnosti od EEG signalov brezžično aktivira proizvajanje pulzov NIR svetlobe v podkožnem vsadku.

Brezžično napajani optogenetski vsadki predstavljajo modularen stik elektronike z živimi celicami, ki omogoča neposreden nadzor nad izražanjem genov preko električnih naprav. Pristop, ki je opisan v seminarju pa je stik možganske aktivnosti z elektrogenetsko napravo. Predstavlja nove možnosti načrtovanja elektromehanskih vsadkov, kot so srčni spodbujevalniki, očesne proteze in inzulinske mikročrpalke.

MBT seminarji 2015

2015-06-04T15:02:01Z

Luka Smole:

'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2014/15'''

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).

Način vnosa:

# The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops & 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
(slovenski naslov povežite z novo stranjo, na kateri bo povzetek)

'''Naslovi odobrenih člankov po temah:'''

'''Gensko spremenjene rastline''' 
# 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
#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

'''Gensko spremenjene živali''' 
# [[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)
# 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

'''Okolje''' 
# 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
# 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

'''Terapevtiki''' 
# 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
# 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

'''Encimi''' 
# 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
# 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
# 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

'''Protitelesa''' 
# 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
# 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
# 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

'''Cepiva''' 
# 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
# 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 "priming-boosting" strategija za imunsko posredovanje pri raku materničnega vratu]]. Anita Kustec, 8. maja 2015
# 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

'''Male molekule in polimeri''' 
# 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
# Purification and characterization of gamma poly glutamic acid from newly Bacillus licheniformis NRC20; http://www.sciencedirect.com/science/article/pii/S0141813014008216. Uroš Stupar, 14. maja 2015
# 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
# 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

'''Pretvorba biomase''' 
# 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
# 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
# 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

'''Metabolično inženirstvo''' 
# 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
# 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
# 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

'''Biološki viri energije''' 
# 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
# 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
# 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
# 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

'''Novi pristopi v molekularni biotehnologiji''' 
# Exploring the potential of algae/bacteria interactions; http://www.sciencedirect.com/science/article/pii/S0958166915000269. Matja Zalar, 11. junija
# 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. Monika Škrjanc, 11. junija
# 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

MBT seminarji 2015

2015-03-09T08:21:38Z

Luka Smole:

RNA-guided human genome engineering via Cas9

2015-01-13T08:10:44Z

Luka Smole:

Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., Church, G.M. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/pdf/nihms471334.pdf RNA-guided human genome engineering via Cas9]. Science 339, 823–6. 2013

Luka Smole

[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F1/ Results figure 1] 
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F2/ Results figure 2] 
[http://www.sciencemag.org/content/suppl/2013/01/03/science.1232033.DC1/Mali.SM.pdf Supplementary Materials]

==Introduction==

In this seminar I will write about CRISPR/Cas system, in this case constructed for human genome engineering. In this research authors engineered bacterial type II CRISPR system to target endogenous AAVS1 locus in human cells with two different sgRNAs to promote homologous recombination. The article I will focus on was published in Science by Prashant Mali from George M. Church’s research group from Harvard Medical School in Boston, Department of Genetics in 2013. George M. Church is one of the most cited and well respected scientists in the field of synthetic biology. Since 2013, his research group published many papers on CRISPR/Cas system in highly respected journals such as Nature biotechnology, Nucleic acids research and Science.

*Note:
While writing this seminar, I put a lot of effort in summarizing this research in most comprehensible manner. To read this seminar in less confusing way, I suggest opening the links to figures at the very beginning of reading to better understand design of experiments and results (all important results are summarized in two figures).

==CRISPR/Cas9 system==

Making specific changes in DNA, such as changing, inserting or deleting sequences that code for proteins allows us to engineer cells, tissues and organisms for therapeutic and practical applications. Until now, such genome engineering has required the design and production of proteins with the ability to recognize a specific DNA sequence, such as zinc fingers and TALE (transcription activator like effector) proteins. Such techniques are relatively time consuming and expensive (especially on large scale, such as engineering for therapeutic applications). Thus, research of alternative strategies for triggering site-specific DNA cleavage in eukaryotic cells was in great interest. The bacterial protein, Cas9, had the potential to enable a simpler approach to genome engineering. It is a DNA-cleaving enzyme that can be programmed with single guiding RNA molecules (sgRNA) to recognize specific DNA sequences. This way, there is no need to engineer a new protein for each new DNA target sequence.<ref name="ref1">Sternberg, S.H., Redding, S., Jinek, M., Greene, E.C., Doudna, J. a, 2014. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–7. doi:10.1038/nature13011</ref>.
This single RNA–single protein CRISPR system is derived from a natural adaptive immune system in bacteria and archaea. Prokaryotes have evolved diverse RNA-mediated systems that use short CRISPR RNAs (crRNAs) and Cas (CRISPR-associated) proteins to detect and defend against foreign DNA, such as phage DNA. Bacteria harbouring CRISPR/Cas loci respond to viral and plasmid challenge by integrating short fragments of the foreign nucleic acid (protospacers) into the host chromosome at one end of the CRISPR locus. The transcript of CRISPR loci is short CRISPR RNAs (crRNAs) that direct Cas protein-mediated cleavage of complementary target sequences within invading foreign (viral or plasmid) DNA. In type II CRISPR/Cas systems, Cas9 functions as a RNA-guided endonuclease that uses a dual-guide RNA. Guide RNA consists of crRNA (which interacts with Cas9 protein by “handle”) and trans-activating crRNA (tracrRNA) for target recognition and cleavage by a mechanism involving two nuclease active sites that together generate doublestranded DNA breaks (DSB). For schematic representation of CRISPR/Cas system, see this figure: [http://2013.igem.org/wiki/images/9/9f/CRISPR.png].
As stated above, zinc fingers and TALEs are powerful tools in synthetic biology, but there are some drawbacks, because it remains time consuming and expensive to develop large-scale protein libraries for genome interrogation<ref name="ref5">Gilbert, L. a, Larson, M.H., Morsut, L., Liu, Z., Brar, G. a, Torres, S.E., Stern-Ginossar, N., Brandman, O., Whitehead, E.H., Doudna, J. a, Lim, W. a, Weissman, J.S., Qi, L.S., 2013. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–51. doi:10.1016/j.cell.2013.06.044</ref>.
Note that authors use different terms for guiding RNAs in CRISPR systems due to lack of established terminology on this relatively new field of synthetic biology. So far the popular terms are short-guide and single-guide RNA, but they mean the same RNA that “guides” Cas9 nuclease to target DNA sequence. Some authors refer crRNA/tracrRNA complex as single-guide RNA or sgRNA (because it works for both Cas9 binding and DNA target site recognition as single transcript).

==Homologous recombination (HR), non-homologous end joining (NHEJ)==

It is important to be familiar with mechanisms of homologous recombination (HR) and non-homologous end joining (NHEJ) for understanding the design and principle of this human genome engineering study. We must point out that HR is a process that uses a desired homologous repair “primer” of donor DNA as a template from which it copies the information, which was lost during DSB. NHEJ on the other hand simply joins ends without homology and often results in deletions and/or insertions. These mechanisms are well shown: [http://www.nature.com/scitable/content/repair-of-dna-double-strand-breaks-by-41523 here]<ref name="ref4">Jeggo, P. a, Löbrich, M., 2007. DNA double-strand breaks: their cellular and clinical impact? Oncogene 26, 7717–9. doi:10.1038/sj.onc.1210868 </ref>.

==Design of CRISPR/Cas9 system for RNA-guided human genome engineering==

In this research 1 engineered bacterial type II CRISPR system to target endogenous AAVS1 locus in human cells with two different sgRNAs to promote homologous recombination. They synthesized a human codon-optimized version of the Cas9 protein bearing a C terminus SV40 nuclear localization signal and cloned it into a mammalian expression system. Here is a plasmid map of this construct: https://www.addgene.org/41815/.
To direct Cas9 to specific sequences of interest, single guide RNAs (sgRNAs) was expressed from the human U6 polymerase III promoter. Schematic representation of construct designs is shown of figure 1 A: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F1/]. The first important constrain is that U6 transcription must initiate with G. The second constrain in all CRISPR/Cas systems is the requirement for the PAM (protospacer-adjacent motif) sequence -NGG following the ≈20 bp sgRNA target. Regarding the mentioned facts, CRISPR/Cas9 system can in principle target any genomic site of the form G(N)20GG.
They developed a GFP reporter assay to test the functionality CRISPR/Cas9 system as genome engineering tool. To test the efficiency of system at stimulating HR, two sgRNAs (T1 and T2) that target the intervening AAVS1 fragment were constructed (Figure 1 B [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F1/]).
A genomically integrated GFP coding sequence is disrupted by the insertion of a stop codon and a 68bp genomic fragment from the AAVS1 locus. Restoration of the GFP sequence by homologous recombination with an appropriate donor sequence results in GFP+ cells enabling quantification by FACS (flow activated cell sorting). HR stimulation rate was then compared to TAL effector nuclease heterodimer (TALEN) targeting the same region.

==Results==

===Targeting GFP reporter system===
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F1/ Figure 1]

Successful HR events were observed when targeting previously described GFP reporter system. Gene correction rates were 3% when T1 sgRNA and 8% when T2 sgRNAs was used in CRISPR/Cas9 system.
This system has proved to be more rapid than TALENs with the first GFP+ cells appearing ~20 hours post transfection while ~40 hours for the TALENs.
HR was observed only when introduction all three components of CRISPR/Cas9 system were present (repair donor, Cas9 protein, and gRNA). This result confirms that all components are required for genome editing.
When mutating the target genomic site, sgRNA had no effect at HR in that locus, demonstrating that CRISPR/Cas9 mediated genome editing is sequence specific.
293T cells transfection with various combinations of constructs (humanized Cas9+T1 sgRNA and humanized Cas9+T2 sgRNA). NHEJ rates measurement (4 days after nucleofection) was performed by deep sequencing, detecting genomic deletions and insertions at DSBs. 293T targeting by both sgRNAs is efficient (10-24%) and sequence specific. These results show that using T2 sgRNA yelds higher genome targeting rates. These result might be the consequence of local target DNA structure, due to better T2 sgRNA affinity of first 12 bp after PAM sequence. Jinek ''et al''<ref name="ref3">Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., Doudna, J., 2013. RNA-programmed genome editing in human cells. Elife 2, e00471. doi:10.7554/eLife.00471.</ref> suggested that target sites must perfectly match the PAM sequence NGG and the following 8-12 base at the 3′ end of the gRNA.

===Targeting in native endogenous AAVS1 locus===
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F2/ Figure 2]

After successful targeting of integrated reporter, the next goal was to modify a native locus. SgRNAs to target the AAVS1 locus were used (described above in paragraph Design of CRISPR/Cas9 system for RNA-guided human genome engineering). AAVS1 locus is located in the PPP1R12C gene on chromosome 19, which is ubiquitously expressed across most tissues. Genome modification tests were performed in 293T, K562, and PGP1 human iPS cells. Results were analyzed by next-generation sequencing of the targeted locus.
As in previous experiments of targeting the GFP reporter assay, authors have observed high rates of NHEJ at the endogenous locus for all three cell types. The two gRNAs, T1 and T2, achieved NHEJ rates of 10 and 25% in 293Ts, 13 and 38% in K562s, and 2 and 4% in PGP1-iPS cells, respectively (Fig. 2B). As observed, NHEJ rates vary in cell types, most probably due to different complex endogenous processes. As seen on figures, total count and location of deletions caused by NHEJ for T1 and T2 were centered around the target site positions. These results clearly show, once more, the sequence specificity CRISPR/Cas9 system.
Simultaneous introduction of both T1 and T2 gRNAs resulted in high efficiency deletion of the targeted 19bp fragment, demonstrating that multiplexed editing of genomic loci is feasible using this approach.

===Modifying the native AAVS1 locus===

Using CRISPR/Cas9 system to induce HR for integration of donor construct or an oligo donor into the endogenous loci in human cells has a great potential for future therapeutic use. Authors confirmed HR-mediated integration in the native AAVS1 locus using both approaches by PCR and Sanger sequencing. PCR screen [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F2/ (see Figure 2C)] confirmed that 21/24 randomly picked 293T clones were successfully targeted. Similar PCR screen confirmed 3/7 randomly picked29 PGP1‐iPS clones were also successfully targeted. Also, short 90-mer oligos could also effect robust targeting at the endogenous AAVS1 locus. In [http://www.sciencemag.org/content/suppl/2013/01/03/science.1232033.DC1/Mali.SM.pdf Supplementary Materials, Figure S10] we can se that NHEJ rate was 38%.

===Bioinformatically generated gRNA-targetable sequences===

This versatile RNA-guided genome engineerig system can be adapted to modify other genomic sites just by modifying the sequence of sgRNA expression vector used to match a compatible sequence in the locus of interest. To facilitate this process, authors bioinformatically generated ~190,000 specifically gRNA-targetable sequences targeting ~40.5% exons of genes in the human genome (shown in [http://www.sciencemag.org/content/suppl/2013/01/03/science.1232033.DC1/Mali.SM.pdf Supplementary Materials Table S1]). These target sequences are incorporatedinto a 200bp format compatible with multiplex synthesis on DNA arrays (shown in [http://www.sciencemag.org/content/suppl/2013/01/03/science.1232033.DC1/Mali.SM.pdf Supplementary Materials], Figure S11 and tables S2 and S3). This work provides a ready genome-wide source of potential target sites in the human genome and a methodology for multiplex gRNA synthesis.

==Other Cas9 prospects==

Recent studies have shown possibilities of versatile Cas9 mediated use for genome editing and regulation. Great versatility and potential of the Cas9 as combining factor with ability to bring together DNA, RNA and proteins. Proteins can be targeted to any dsDNA sequence by simply fusing them to a nuclease-null Cas9 and expressing a suitable sgRNA. Note that some authors use “dead” Cas9 (dCas9) as term for Cas9 protein with lack of nuclease activity. Consequently, Cas9 can bring any fusion protein together with any fusion RNA at any dsDNA sequence by covalent attachment to dCas9 or to sgRNAs, or by noncovalent binding to covalently attached molecules. Knowing that effective concentration is important in regulation of biological processes, CRISPR/Cas9 system can be used as a single unifying factor, capable of mediating biologic interactions. Therefore, it has a great potential for use in investigating and engineering living systems.
For example, transcription is dependent on the assembly of regulatory complexes and their interactions with chromatin. By targeting dCas9 to important binding sites for transcription factors, it should be possible to obstruct the binding of these factors and thereby exclude their role in transcription. Similarly, individual factors with unknown roles could be selectively recruited to almost any desired sequence by dCas9 fusions or sgRNA tethers with only slightly less precision. Together, these capabilities may allow a component-by-component approach to perturbing endogenous gene regulation<ref name="ref7">Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–63 (2013)</ref>.

===Transcriptional activation===

For engineering purposes, it is often useful to directly upregulate the transcription of endogenous genes to a desired level of activity. Experiments with zinc finger effectors and transcription activator–like (TAL) effectors demonstrated that multiple VP64 activator domains localized 5′ of the transcription start site yield synergistic effects. It was shown that Cas9-mediated localization functions similarly with dCas9–VP64. It is important to know that the rate of activation can vary among targeted genes. It requires synergy between multiple Cas9-sgRNA activators for robust transcription. Activation is probably dependent on local chromatin structure, unique interactions with endogenous transcriptional machinery and the Cas9 biochemistry. Elucidation of these effects as well as evaluation of additional Cas9 orthologs will be necessary for fine tuning of control over endogenous transcription.
The capability to upregulate any endogenous gene or combination of genes in trans-acting manner has tremendous implications for ability to investigate and control cellular behavior. In particular, multiplexed sgRNA libraries targeting every known gene could help point out the factors responsible for important cellular processes, such as differentiation.

===Transcriptional repression===

Fusing of repressor domains to zinc finger effector or TAL effector proteins potently suppresses endogenous transcription. By using a similar architecture for dCas9–KRAB or related fusion proteins or sgRNA-based tethers, it should be possible to repress genes with equivalent efficacy and far greater ease of targeting. Indeed, a dCas9–KRAB fusion has been recently shown to induce modest repression using single guide RNAs. Localizing additional repressors and optimizing the structure of the fusion protein could greatly increase the potency of this approach. The ability to repress transcription will not only complement studies using transcriptional activation, but may also be useful for antiviral applications in eukaryotic cells. By preventing the transcription of invading viral genomes, Cas9 repressors could in principle “equip” a transgenic organism with immune to many DNA viruses, targeted with sufficient sgRNAs. This might be a great advantage for crops and domesticated animals.

===Improving specificity===

An increasingly recognized limitation in Cas9-mediated genome engineering applications is their specificity of targeting. The sgRNA-Cas9 complexes are in general tolerant of 1–3 mismatches in their target and occasionally more. It depends on the function of the Cas9 ortholog, the sgRNA architecture, the targeted sequence, the PAM, and also the relative dose and duration of these reagents. Although imperfect Cas9 specificity is a major reason for concern, there are several methods of potentially improving this drawback. Improvements include requiring multiple sgRNA-Cas9 complexes for activity, reducing affinity while increasing cooperativity, establishing competition between inactive and active forms, discovering improved natural orthologs, engineering improved variants and choosing targeting sgRNAs wisely.

===Engineering Cas9-targeted recombinases===

Despite the effectiveness of nuclease-based methods in editing genomes, safe in vivo gene correction in humans remains difficult. Most notably, the introduction of a double-strand break or even a nick at the wrong off-target site can lead to unexpected mutations or rearrangements that may have consequences in carcinogenesis. Site-specific recombinase, and potentially transposase enzymes present fewer problems by tightly controlling generation of DSBs to coordinate donor-target coupling. By fusing the catalytic domain of a small serine recombinase to Cas9, analogous to previous zinc finger and TAL fusions, it may be possible to create an RNA-guided recombinase enzyme. Because the activity of such retargeted fusion recombinases is generally low, extensive directed evolution may be necessary to produce a useful RNA-guided recombinase.

===Discovering or evolving improved Cas9 proteins===

It is possible that certain Cas9 orthologs might prove more specific than the Cas9 from S. pyogenes. It is unlikely that Cas9 proteins with longer PAM requirements will exhibit greater overall specificity, as the selective pressure for accurate recognition of the combined spacer and PAM remains constant. However, Cas9 proteins from species with larger genomes may be somewhat more specific, and those that have undergone frequent horizontal gene transfer along with their CRISPR loci and consequently been selected for avoidance of multiple host genomes are likely the most specific of all.
The best Cas9 proteins identified in nature might be improved by rational design (usually by mutagenesis studies), directed evolution or ideally a combination of the two. One attractive strategy for improving specificity is to reduce the basal Cas9 affinity for DNA, which could be dimminished at target sites by employing two cooperatively binding sgRNAs with complementary 3′ overhangs that target adjacent protospacers. Alternatively, the PAM might be changed to expand the range of targetable sites or enlarged to increase specificity, although such alteration may not be accessible by rational design alone.
PAM alteration and more complex modifications might be accessible using directed evolution, including increasing the overall specificity of each Cas9 monomer. Such experiments must be designed to select for activity at a perfectly matched protospacer. Activity at mismatched sites, preferably those identified as problematic by specificity measurement assays is also important. Ideally, the process would result in selection against many mismatched protospacers at any one time, and the process would be repeated over many rounds of selection. Methods of directed evolution would be convenient for this challenge<ref name="ref7">Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–63 (2013)</ref>..

==Conclusion==

These results show that RNA-programmed genome editing is a straightforward strategy for introducing site-specific genetic changes in human cells. Due to ease of design and effectiveness, in 2013 CRISPR/Cas system took over most the researcher’s attention in field of tools for genome regulation and modification. It seems that it is likely to become competitive with existing approaches based on zinc finger nucleases and transcription activator-like effector nucleases, and could lead to a new generation of experiments in the field of genome engineering for such complex genomes as human<ref name="ref3">Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., Doudna, J., 2013. RNA-programmed genome editing in human cells. Elife 2, e00471. doi:10.7554/eLife.00471.</ref>
Research into the CRISPR-Cas gene editing system continues at great speed. The ease, low cost and speed of designing an RNA guided endonuclease against a DNA target of interest has caught the imagination of worldwide researchers. In beginning of 2014, the crystal structure of ''Streptococcus pyogenes'' Cas9 was published <ref name="ref6">Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–49 (2014)</ref>. This achievement offers the possibility of rational engineering of this RNA-protein complex based on structural information for the first time.
The interest is not in academic sphere; several startups have been created around the technology. Also, reagent companies are already desinging CRISPR reagents for the research community. A few already commercially available products are CRISPR online design tools, CRISPR paired nickases for high specificity genome editing and Cas9 mRNA and expression plasmids.<ref name="ref2">Baker, M., 2014. Gene editing at CRISPR speed. Nat. Biotechnol. 32, 309–12. doi:10.1038/nbt.2863</ref>.

==References==

<references />

[[SB students resources]]

RNA-guided human genome engineering via Cas9

2015-01-12T17:49:29Z

Luka Smole:

Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., Church, G.M. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/pdf/nihms471334.pdf RNA-guided human genome engineering via Cas9]. Science 339, 823–6. 2013

Luka Smole

[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F1/ Results figure 1] 
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F2/ Results figure 2] 
[http://www.sciencemag.org/content/suppl/2013/01/03/science.1232033.DC1/Mali.SM.pdf Supplementary Materials]

==Introduction==

In this seminar I will write about CRISPR/Cas system, in this case constructed for human genome engineering. In this research authors engineered bacterial type II CRISPR system to target endogenous AAVS1 locus in human cells with two different sgRNAs to promote homologous recombination. The article I will focus on was published in Science by Prashant Mali from George M. Church’s research group from Harvard Medical School in Boston, Department of Genetics in 2013. George M. Church is one of most cited and well respected scientists in field of synthetic biology. Since 2013, his research group published many papers on CRISPR/Cas system in highly respected journals such as Nature biotechnology, Nucleic acids research and Science.

*Note:
While writing this seminar, I put a lot of effort in summarizing this research in most comprehensible manner. To read this seminar in less confusing way, I suggest opening the links to figures at the very beginning of reading to better understand design of experiments and results (all important results are summarized in two figures).

==CRISPR/Cas9 system==

Making specific changes in DNA, such as changing, inserting or deleting sequences that code for proteins allows us to engineer cells, tissues and organisms for therapeutic and practical applications. Until now, such genome engineering has required the design and production of proteins with the ability to recognize a specific DNA sequence, such as zinc fingers and TALE (transcription activator like effector) proteins. Such techniques are relatively time consuming and expensive (especially on large scale, such as engineering for therapeutic applications). Thus, research of alternative strategies for triggering site-specific DNA cleavage in eukaryotic cells was in great interest. The bacterial protein, Cas9, had the potential to enable a simpler approach to genome engineering. It is a DNA-cleaving enzyme that can be programmed with single guiding RNA molecules (sgRNA) to recognize specific DNA sequences. This way, there is no need to engineer a new protein for each new DNA target sequence.<ref name="ref1">Sternberg, S.H., Redding, S., Jinek, M., Greene, E.C., Doudna, J. a, 2014. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–7. doi:10.1038/nature13011</ref>.
This single RNA–single protein CRISPR system is derived from a natural adaptive immune system in bacteria and archaea. Prokaryotes have evolved diverse RNA-mediated systems that use short CRISPR RNAs (crRNAs) and Cas (CRISPR-associated) proteins to detect and defend against foreign DNA, such as phage DNA. Bacteria harbouring CRISPR/Cas loci respond to viral and plasmid challenge by integrating short fragments of the foreign nucleic acid (protospacers) into the host chromosome at one end of the CRISPR locus. The transcript of CRISPR loci is short CRISPR RNAs (crRNAs) that direct Cas protein-mediated cleavage of complementary target sequences within invading foreign (viral or plasmid) DNA. In type II CRISPR/Cas systems, Cas9 functions as a RNA-guided endonuclease that uses a dual-guide RNA. Guide RNA consists of crRNA (which interacts with Cas9 protein by “handle”) and trans-activating crRNA (tracrRNA) for target recognition and cleavage by a mechanism involving two nuclease active sites that together generate doublestranded DNA breaks (DSB). For schematic representation of CRISPR/Cas system, see this figure: [http://2013.igem.org/wiki/images/9/9f/CRISPR.png].
As stated above, zinc fingers and TALEs are powerful tools in synthetic biology, but there are some drawbacks, because it remains time consuming and expensive to develop large-scale protein libraries for genome interrogation<ref name="ref5">Gilbert, L. a, Larson, M.H., Morsut, L., Liu, Z., Brar, G. a, Torres, S.E., Stern-Ginossar, N., Brandman, O., Whitehead, E.H., Doudna, J. a, Lim, W. a, Weissman, J.S., Qi, L.S., 2013. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–51. doi:10.1016/j.cell.2013.06.044</ref>.
Note that authors use different terms for guiding RNAs in CRISPR systems due to lack of established terminology on this relatively new field of synthetic biology. So far the popular terms are short-guide and single-guide RNA, but they mean the same RNA that “guides” Cas9 nuclease to target DNA sequence. Some authors refer crRNA/tracrRNA complex as single-guide RNA or sgRNA (because it works for both Cas9 binding and DNA target site recognition as single transcript).

==Homologous recombination (HR), non-homologous end joining (NHEJ)==

It is important to be familiar with mechanisms of homologous recombination (HR) and non-homologous end joining (NHEJ) for understanding the design and principle of this human genome engineering study. We must point out that HR is a process that uses a desired homologous repair “primer” of donor DNA as a template from which it copies the information, which was lost during DSB. NHEJ on the other hand simply joins ends without homology and often results in deletions and/or insertions. These mechanisms are well shown: [http://www.nature.com/scitable/content/repair-of-dna-double-strand-breaks-by-41523 here]<ref name="ref4">Jeggo, P. a, Löbrich, M., 2007. DNA double-strand breaks: their cellular and clinical impact? Oncogene 26, 7717–9. doi:10.1038/sj.onc.1210868 </ref>.

==Design of CRISPR/Cas9 system for RNA-guided human genome engineering==

In this research 1 engineered bacterial type II CRISPR system to target endogenous AAVS1 locus in human cells with two different sgRNAs to promote homologous recombination. They synthesized a human codon-optimized version of the Cas9 protein bearing a C terminus SV40 nuclear localization signal and cloned it into a mammalian expression system. Here is a plasmid map of this construct: https://www.addgene.org/41815/.
To direct Cas9 to specific sequences of interest, single guide RNAs (sgRNAs) was expressed from the human U6 polymerase III promoter. Schematic representation of construct designs is shown of figure 1 A: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F1/]. The first important constrain is that U6 transcription must initiate with G. The second constrain in all CRISPR/Cas systems is the requirement for the PAM (protospacer-adjacent motif) sequence -NGG following the ≈20 bp sgRNA target. Regarding the mentioned facts, CRISPR/Cas9 system can in principle target any genomic site of the form G(N)20GG.
They developed a GFP reporter assay to test the functionality CRISPR/Cas9 system as genome engineering tool. To test the efficiency of system at stimulating HR, two sgRNAs (T1 and T2) that target the intervening AAVS1 fragment were constructed (Figure 1 B [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F1/]).
A genomically integrated GFP coding sequence is disrupted by the insertion of a stop codon and a 68bp genomic fragment from the AAVS1 locus. Restoration of the GFP sequence by homologous recombination with an appropriate donor sequence results in GFP+ cells enabling quantification by FACS (flow activated cell sorting). HR stimulation rate was then compared to TAL effector nuclease heterodimer (TALEN) targeting the same region.

==Results==

===Targeting GFP reporter system===
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F1/ Figure 1]

Successful HR events were observed when targeting previously described GFP reporter system. Gene correction rates were 3% when T1 sgRNA and 8% when T2 sgRNAs was used in CRISPR/Cas9 system.
This system has proved to be more rapid than TALENs with the first GFP+ cells appearing ~20 hours post transfection while ~40 hours for the TALENs.
HR was observed only when introduction all three components of CRISPR/Cas9 system were present (repair donor, Cas9 protein, and gRNA). This result confirms that all components are required for genome editing.
When mutating the target genomic site, sgRNA had no effect at HR in that locus, demonstrating that CRISPR/Cas9 mediated genome editing is sequence specific.
293T cells transfection with various combinations of constructs (humanized Cas9+T1 sgRNA and humanized Cas9+T2 sgRNA). NHEJ rates measurement (4 days after nucleofection) was performed by deep sequencing, detecting genomic deletions and insertions at DSBs. 293T targeting by both sgRNAs is efficient (10-24%) and sequence specific. These results show that using T2 sgRNA yelds higher genome targeting rates. These result might be the consequence of local target DNA structure, due to better T2 sgRNA affinity of first 12 bp after PAM sequence. Jinek ''et al''<ref name="ref3">Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., Doudna, J., 2013. RNA-programmed genome editing in human cells. Elife 2, e00471. doi:10.7554/eLife.00471.</ref> suggested that target sites must perfectly match the PAM sequence NGG and the following 8-12 base at the 3′ end of the gRNA.

===Targeting in native endogenous AAVS1 locus===
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F2/ Figure 2]

After successful targeting of integrated reporter, the next goal was to modify a native locus. SgRNAs to target the AAVS1 locus were used (described above in paragraph Design of CRISPR/Cas9 system for RNA-guided human genome engineering). AAVS1 locus is located in the PPP1R12C gene on chromosome 19, which is ubiquitously expressed across most tissues. Genome modification tests were performed in 293T, K562, and PGP1 human iPS cells. Results were analyzed by next-generation sequencing of the targeted locus.
As in previous experiments of targeting the GFP reporter assay, authors have observed high rates of NHEJ at the endogenous locus for all three cell types. The two gRNAs, T1 and T2, achieved NHEJ rates of 10 and 25% in 293Ts, 13 and 38% in K562s, and 2 and 4% in PGP1-iPS cells, respectively (Fig. 2B). As observed, NHEJ rates vary in cell types, most probably due to different complex endogenous processes. As seen on figures, total count and location of deletions caused by NHEJ for T1 and T2 were centered around the target site positions. These results clearly show, once more, the sequence specificity CRISPR/Cas9 system.
Simultaneous introduction of both T1 and T2 gRNAs resulted in high efficiency deletion of the targeted 19bp fragment, demonstrating that multiplexed editing of genomic loci is feasible using this approach.

===Modifying the native AAVS1 locus===

Using CRISPR/Cas9 system to induce HR for integration of donor construct or an oligo donor into the endogenous loci in human cells has a great potential for future therapeutic use. Authors confirmed HR-mediated integration in the native AAVS1 locus using both approaches by PCR and Sanger sequencing. PCR screen [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F2/ (see Figure 2C)] confirmed that 21/24 randomly picked 293T clones were successfully targeted. Similar PCR screen confirmed 3/7 randomly picked29 PGP1‐iPS clones were also successfully targeted. Also, short 90-mer oligos could also effect robust targeting at the endogenous AAVS1 locus. In [http://www.sciencemag.org/content/suppl/2013/01/03/science.1232033.DC1/Mali.SM.pdf Supplementary Materials, Figure S10] we can se that NHEJ rate was 38%.

===Bioinformatically generated gRNA-targetable sequences===

This versatile RNA-guided genome engineerig system can be adapted to modify other genomic sites just by modifying the sequence of sgRNA expression vector used to match a compatible sequence in the locus of interest. To facilitate this process, authors bioinformatically generated ~190,000 specifically gRNA-targetable sequences targeting ~40.5% exons of genes in the human genome (shown in [http://www.sciencemag.org/content/suppl/2013/01/03/science.1232033.DC1/Mali.SM.pdf Supplementary Materials Table S1]). These target sequences are incorporatedinto a 200bp format compatible with multiplex synthesis on DNA arrays (shown in [http://www.sciencemag.org/content/suppl/2013/01/03/science.1232033.DC1/Mali.SM.pdf Supplementary Materials], Figure S11 and tables S2 and S3). This work provides a ready genome-wide source of potential target sites in the human genome and a methodology for multiplex gRNA synthesis.

==Other Cas9 prospects==

Recent studies have shown possibilities of versatile Cas9 mediated use for genome editing and regulation. Great versatility and potential of the Cas9 as combining factor with ability to bring together DNA, RNA and proteins. Proteins can be targeted to any dsDNA sequence by simply fusing them to a nuclease-null Cas9 and expressing a suitable sgRNA. Note that some authors use “dead” Cas9 (dCas9) as term for Cas9 protein with lack of nuclease activity. Consequently, Cas9 can bring any fusion protein together with any fusion RNA at any dsDNA sequence by covalent attachment to dCas9 or to sgRNAs, or by noncovalent binding to covalently attached molecules. Knowing that effective concentration is important in regulation of biological processes, CRISPR/Cas9 system can be used as a single unifying factor, capable of mediating biologic interactions. Therefore, it has a great potential for use in investigating and engineering living systems.
For example, transcription is dependent on the assembly of regulatory complexes and their interactions with chromatin. By targeting dCas9 to important binding sites for transcription factors, it should be possible to obstruct the binding of these factors and thereby exclude their role in transcription. Similarly, individual factors with unknown roles could be selectively recruited to almost any desired sequence by dCas9 fusions or sgRNA tethers with only slightly less precision. Together, these capabilities may allow a component-by-component approach to perturbing endogenous gene regulation<ref name="ref7">Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–63 (2013)</ref>.

===Transcriptional activation===

For engineering purposes, it is often useful to directly upregulate the transcription of endogenous genes to a desired level of activity. Experiments with zinc finger effectors and transcription activator–like (TAL) effectors demonstrated that multiple VP64 activator domains localized 5′ of the transcription start site yield synergistic effects. It was shown that Cas9-mediated localization functions similarly with dCas9–VP64. It is important to know that the rate of activation can vary among targeted genes. It requires synergy between multiple Cas9-sgRNA activators for robust transcription. Activation is probably dependent on local chromatin structure, unique interactions with endogenous transcriptional machinery and the Cas9 biochemistry. Elucidation of these effects as well as evaluation of additional Cas9 orthologs will be necessary for fine tuning of control over endogenous transcription.
The capability to upregulate any endogenous gene or combination of genes in trans-acting manner has tremendous implications for ability to investigate and control cellular behavior. In particular, multiplexed sgRNA libraries targeting every known gene could help point out the factors responsible for important cellular processes, such as differentiation.

===Transcriptional repression===

Fusing of repressor domains to zinc finger effector or TAL effector proteins potently suppresses endogenous transcription. By using a similar architecture for dCas9–KRAB or related fusion proteins or sgRNA-based tethers, it should be possible to repress genes with equivalent efficacy and far greater ease of targeting. Indeed, a dCas9–KRAB fusion has been recently shown to induce modest repression using single guide RNAs. Localizing additional repressors and optimizing the structure of the fusion protein could greatly increase the potency of this approach. The ability to repress transcription will not only complement studies using transcriptional activation, but may also be useful for antiviral applications in eukaryotic cells. By preventing the transcription of invading viral genomes, Cas9 repressors could in principle “equip” a transgenic organism with immune to many DNA viruses, targeted with sufficient sgRNAs. This might be a great advantage for crops and domesticated animals.

===Improving specificity===

An increasingly recognized limitation in Cas9-mediated genome engineering applications is their specificity of targeting. The sgRNA-Cas9 complexes are in general tolerant of 1–3 mismatches in their target and occasionally more. It depends on the function of the Cas9 ortholog, the sgRNA architecture, the targeted sequence, the PAM, and also the relative dose and duration of these reagents. Although imperfect Cas9 specificity is a major reason for concern, there are several methods of potentially improving this drawback. Improvements include requiring multiple sgRNA-Cas9 complexes for activity, reducing affinity while increasing cooperativity, establishing competition between inactive and active forms, discovering improved natural orthologs, engineering improved variants and choosing targeting sgRNAs wisely.

===Engineering Cas9-targeted recombinases===

Despite the effectiveness of nuclease-based methods in editing genomes, safe in vivo gene correction in humans remains difficult. Most notably, the introduction of a double-strand break or even a nick at the wrong off-target site can lead to unexpected mutations or rearrangements that may have consequences in carcinogenesis. Site-specific recombinase, and potentially transposase enzymes present fewer problems by tightly controlling generation of DSBs to coordinate donor-target coupling. By fusing the catalytic domain of a small serine recombinase to Cas9, analogous to previous zinc finger and TAL fusions, it may be possible to create an RNA-guided recombinase enzyme. Because the activity of such retargeted fusion recombinases is generally low, extensive directed evolution may be necessary to produce a useful RNA-guided recombinase.

===Discovering or evolving improved Cas9 proteins===

It is possible that certain Cas9 orthologs might prove more specific than the Cas9 from S. pyogenes. It is unlikely that Cas9 proteins with longer PAM requirements will exhibit greater overall specificity, as the selective pressure for accurate recognition of the combined spacer and PAM remains constant. However, Cas9 proteins from species with larger genomes may be somewhat more specific, and those that have undergone frequent horizontal gene transfer along with their CRISPR loci and consequently been selected for avoidance of multiple host genomes are likely the most specific of all.
The best Cas9 proteins identified in nature might be improved by rational design (usually by mutagenesis studies), directed evolution or ideally a combination of the two. One attractive strategy for improving specificity is to reduce the basal Cas9 affinity for DNA, which could be dimminished at target sites by employing two cooperatively binding sgRNAs with complementary 3′ overhangs that target adjacent protospacers. Alternatively, the PAM might be changed to expand the range of targetable sites or enlarged to increase specificity, although such alteration may not be accessible by rational design alone.
PAM alteration and more complex modifications might be accessible using directed evolution, including increasing the overall specificity of each Cas9 monomer. Such experiments must be designed to select for activity at a perfectly matched protospacer. Activity at mismatched sites, preferably those identified as problematic by specificity measurement assays is also important. Ideally, the process would result in selection against many mismatched protospacers at any one time, and the process would be repeated over many rounds of selection. Methods of directed evolution would be convenient for this challenge<ref name="ref7">Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–63 (2013)</ref>..

==Conclusion==

These results show that RNA-programmed genome editing is a straightforward strategy for introducing site-specific genetic changes in human cells. Due to ease of design and effectiveness, in 2013 CRISPR/Cas system took over most the researcher’s attention in field of tools for genome regulation and modification. It seems that it is likely to become competitive with existing approaches based on zinc finger nucleases and transcription activator-like effector nucleases, and could lead to a new generation of experiments in the field of genome engineering for such complex genomes as human<ref name="ref3">Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., Doudna, J., 2013. RNA-programmed genome editing in human cells. Elife 2, e00471. doi:10.7554/eLife.00471.</ref>
Research into the CRISPR-Cas gene editing system continues at great speed. The ease, low cost and speed of designing an RNA guided endonuclease against a DNA target of interest has caught the imagination of worldwide researchers. In beginning of 2014, the crystal structure of ''Streptococcus pyogenes'' Cas9 was published <ref name="ref6">Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–49 (2014)</ref>. This achievement offers the possibility of rational engineering of this RNA-protein complex based on structural information for the first time.
The interest is not in academic sphere; several startups have been created around the technology. Also, reagent companies are already desinging CRISPR reagents for the research community. A few already commercially available products are CRISPR online design tools, CRISPR paired nickases for high specificity genome editing and Cas9 mRNA and expression plasmids.<ref name="ref2">Baker, M., 2014. Gene editing at CRISPR speed. Nat. Biotechnol. 32, 309–12. doi:10.1038/nbt.2863</ref>.

==References==

<references />

[[SB students resources]]

RNA-guided human genome engineering via Cas9

2015-01-12T17:47:04Z

Luka Smole:

Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., Church, G.M. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/pdf/nihms471334.pdf RNA-guided human genome engineering via Cas9]. Science 339, 823–6. 2013

Luka Smole

[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F1/ Results figure 1] 
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F2/ Results figure 2] 
[http://www.sciencemag.org/content/suppl/2013/01/03/science.1232033.DC1/Mali.SM.pdf Supplementary Materials]

==Introduction==

In this seminar I will write about CRISPR/Cas system, in this case constructed for human genome engineering. In this research authors engineered bacterial type II CRISPR system to target endogenous AAVS1 locus in human cells with two different sgRNAs to promote homologous recombination. The article I will focus on was published in Science by Prashant Mali from George M. Church’s research group from Harvard Medical School in Boston, Department of Genetics in 2013. George M. Church is one of most cited and well respected scientists in field of synthetic biology. Since 2013, his research group published many papers on CRISPR/Cas system in highly respected journals such as Nature biotechnology, Nucleic acids research and Science.

*Note:
While writing this seminar, I put a lot of effort in summarizing this research in most comprehensible manner. To read this seminar in less confusing way, I suggest opening the links to figures at the very beginning of reading to better understand design of experiments and results (all important results are summarized in two figures).

==CRISPR/Cas9 system==

Making specific changes in DNA, such as changing, inserting or deleting sequences that code for proteins allows us to engineer cells, tissues and organisms for therapeutic and practical applications. Until now, such genome engineering has required the design and production of proteins with the ability to recognize a specific DNA sequence, such as zinc fingers and TALE (transcription activator like effector) proteins. Such techniques are relatively time consuming and expensive (especially on large scale, such as engineering for therapeutic applications). Thus, research of alternative strategies for triggering site-specific DNA cleavage in eukaryotic cells was in great interest. The bacterial protein, Cas9, had the potential to enable a simpler approach to genome engineering. It is a DNA-cleaving enzyme that can be programmed with single guiding RNA molecules (sgRNA) to recognize specific DNA sequences. This way, there is no need to engineer a new protein for each new DNA target sequence.<ref name="ref1">Sternberg, S.H., Redding, S., Jinek, M., Greene, E.C., Doudna, J. a, 2014. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–7. doi:10.1038/nature13011</ref>.
This single RNA–single protein CRISPR system is derived from a natural adaptive immune system in bacteria and archaea. Prokaryotes have evolved diverse RNA-mediated systems that use short CRISPR RNAs (crRNAs) and Cas (CRISPR-associated) proteins to detect and defend against foreign DNA, such as phage DNA. Bacteria harbouring CRISPR/Cas loci respond to viral and plasmid challenge by integrating short fragments of the foreign nucleic acid (protospacers) into the host chromosome at one end of the CRISPR locus. The transcript of CRISPR loci is short CRISPR RNAs (crRNAs) that direct Cas protein-mediated cleavage of complementary target sequences within invading foreign (viral or plasmid) DNA. In type II CRISPR/Cas systems, Cas9 functions as a RNA-guided endonuclease that uses a dual-guide RNA. Guide RNA consists of crRNA (which interacts with Cas9 protein by “handle”) and trans-activating crRNA (tracrRNA) for target recognition and cleavage by a mechanism involving two nuclease active sites that together generate doublestranded DNA breaks (DSB). For schematic representation of CRISPR/Cas system, see this figure: [http://2013.igem.org/wiki/images/9/9f/CRISPR.png].
As stated above, zinc fingers and TALEs are powerful tools in synthetic biology, but there are some drawbacks, because it remains time consuming and expensive to develop large-scale protein libraries for genome interrogation<ref name="ref5">Gilbert, L. a, Larson, M.H., Morsut, L., Liu, Z., Brar, G. a, Torres, S.E., Stern-Ginossar, N., Brandman, O., Whitehead, E.H., Doudna, J. a, Lim, W. a, Weissman, J.S., Qi, L.S., 2013. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–51. doi:10.1016/j.cell.2013.06.044</ref>.
Note that authors use different terms for guiding RNAs in CRISPR systems due to lack of established terminology on this relatively new field of synthetic biology. So far the popular terms are short-guide and single-guide RNA, but they mean the same RNA that “guides” Cas9 nuclease to target DNA sequence. Some authors refer crRNA/tracrRNA complex as single-guide RNA or sgRNA (because it works for both Cas9 binding and DNA target site recognition as single transcript).

==Homologous recombination (HR), non-homologous end joining (NHEJ)==

It is important to be familiar with mechanisms of homologous recombination (HR) and non-homologous end joining (NHEJ) for understanding the design and principle of this human genome engineering study. We must point out that HR is a process that uses a desired homologous repair “primer” of donor DNA as a template from which it copies the information, which was lost during DSB. NHEJ on the other hand simply joins ends without homology and often results in deletions and/or insertions. These mechanisms are well shown: [http://www.nature.com/scitable/content/repair-of-dna-double-strand-breaks-by-41523 here]<ref name="ref4">Jeggo, P. a, Löbrich, M., 2007. DNA double-strand breaks: their cellular and clinical impact? Oncogene 26, 7717–9. doi:10.1038/sj.onc.1210868 </ref>.

==Design of CRISPR/Cas9 system for RNA-guided human genome engineering==

In this research 1 engineered bacterial type II CRISPR system to target endogenous AAVS1 locus in human cells with two different sgRNAs to promote homologous recombination. They synthesized a human codon-optimized version of the Cas9 protein bearing a C terminus SV40 nuclear localization signal and cloned it into a mammalian expression system. Here is a plasmid map of this construct: https://www.addgene.org/41815/.
To direct Cas9 to specific sequences of interest, single guide RNAs (sgRNAs) was expressed from the human U6 polymerase III promoter. Schematic representation of construct designs is shown of figure 1 A: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F1/]. The first important constrain is that U6 transcription must initiate with G. The second constrain in all CRISPR/Cas systems is the requirement for the PAM (protospacer-adjacent motif) sequence -NGG following the ≈20 bp sgRNA target. Regarding the mentioned facts, CRISPR/Cas9 system can in principle target any genomic site of the form G(N)20GG.
They developed a GFP reporter assay to test the functionality CRISPR/Cas9 system as genome engineering tool. To test the efficiency of system at stimulating HR, two sgRNAs (T1 and T2) that target the intervening AAVS1 fragment were constructed (Figure 1 B [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F1/]).
A genomically integrated GFP coding sequence is disrupted by the insertion of a stop codon and a 68bp genomic fragment from the AAVS1 locus. Restoration of the GFP sequence by homologous recombination with an appropriate donor sequence results in GFP+ cells enabling quantification by FACS (flow activated cell sorting). HR stimulation rate was then compared to TAL effector nuclease heterodimer (TALEN) targeting the same region.

==Results==

===Targeting GFP reporter system===
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F1/ Figure 1]

Successful HR events were observed when targeting previously described GFP reporter system. Gene correction rates were 3% when T1 sgRNA and 8% when T2 sgRNAs was used in CRISPR/Cas9 system.
This system has proved to be more rapid than TALENs with the first GFP+ cells appearing ~20 hours post transfection while ~40 hours for the TALENs.
HR was observed only when introduction all three components of CRISPR/Cas9 system were present (repair donor, Cas9 protein, and gRNA). This result confirms that all components are required for genome editing.
When mutating the target genomic site, sgRNA had no effect at HR in that locus, demonstrating that CRISPR/Cas9 mediated genome editing is sequence specific.
293T cells transfection with various combinations of constructs (humanized Cas9+T1 sgRNA and humanized Cas9+T2 sgRNA). NHEJ rates measurement (4 days after nucleofection) was performed by deep sequencing, detecting genomic deletions and insertions at DSBs. 293T targeting by both sgRNAs is efficient (10-24%) and sequence specific. These results show that using T2 sgRNA yelds higher genome targeting rates. These result might be the consequence of local target DNA structure, due to better T2 sgRNA affinity of first 12 bp after PAM sequence. Jinek ''et al''<ref name="ref3">Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., Doudna, J., 2013. RNA-programmed genome editing in human cells. Elife 2, e00471. doi:10.7554/eLife.00471.</ref> suggested that target sites must perfectly match the PAM sequence NGG and the following 8-12 base at the 3′ end of the gRNA.

===Targeting in native endogenous AAVS1 locus===
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F2/ Figure 2]

After successful targeting of integrated reporter, the next goal was to modify a native locus. SgRNAs to target the AAVS1 locus were used (described above in paragraph Design of CRISPR/Cas9 system for RNA-guided human genome engineering). AAVS1 locus is located in the PPP1R12C gene on chromosome 19, which is ubiquitously expressed across most tissues. Genome modification tests were performed in 293T, K562, and PGP1 human iPS cells. Results were analyzed by next-generation sequencing of the targeted locus.
As in previous experiments of targeting the GFP reporter assay, authors have observed high rates of NHEJ at the endogenous locus for all three cell types. The two gRNAs, T1 and T2, achieved NHEJ rates of 10 and 25% in 293Ts, 13 and 38% in K562s, and 2 and 4% in PGP1-iPS cells, respectively (Fig. 2B). As observed, NHEJ rates vary in cell types, most probably due to different complex endogenous processes. As seen on figures, total count and location of deletions caused by NHEJ for T1 and T2 were centered around the target site positions. These results clearly show, once more, the sequence specificity CRISPR/Cas9 system.
Simultaneous introduction of both T1 and T2 gRNAs resulted in high efficiency deletion of the targeted 19bp fragment, demonstrating that multiplexed editing of genomic loci is feasible using this approach.

===Modifying the native AAVS1 locus===

Using CRISPR/Cas9 system to induce HR for integration of donor construct or an oligo donor into the endogenous loci in human cells has a great potential for future therapeutic use. Authors confirmed HR-mediated integration in the native AAVS1 locus using both approaches by PCR and Sanger sequencing. PCR screen [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/figure/F2/ (see Figure 2C)] confirmed that 21/24 randomly picked 293T clones were successfully targeted. Similar PCR screen confirmed 3/7 randomly picked29 PGP1‐iPS clones were also successfully targeted. Also, short 90-mer oligos could also effect robust targeting at the endogenous AAVS1 locus. In [http://www.sciencemag.org/content/suppl/2013/01/03/science.1232033.DC1/Mali.SM.pdf Supplementary Materials, Figure S10] we can se that NHEJ rate was 38%.

===Bioinformatically generated gRNA-targetable sequences===

This versatile RNA-guided genome engineerig system can be adapted to modify other genomic sites just by modifying the sequence of sgRNA expression vector used to match a compatible sequence in the locus of interest. To facilitate this process, authors bioinformatically generated ~190,000 specifically gRNA-targetable sequences targeting ~40.5% exons of genes in the human genome (shown in [http://www.sciencemag.org/content/suppl/2013/01/03/science.1232033.DC1/Mali.SM.pdf Supplementary Materials Table S1]). These target sequences are incorporatedinto a 200bp format compatible with multiplex synthesis on DNA arrays (shown in [http://www.sciencemag.org/content/suppl/2013/01/03/science.1232033.DC1/Mali.SM.pdf Supplementary Materials], Figure S11 and tables S2 and S3). This work provides a ready genome-wide source of potential target sites in the human genome and a methodology for multiplex gRNA synthesis.

==Other Cas9 prospects==

Recent studies have shown possibilities of versatile Cas9 mediated use for genome editing and regulation. Great versatility and potential of the Cas9 as combining factor with ability to bring together DNA, RNA and proteins. Proteins can be targeted to any dsDNA sequence by simply fusing them to a nuclease-null Cas9 and expressing a suitable sgRNA. Note that some authors use “dead” Cas9 (dCas9) as term for Cas9 protein with lack of nuclease activity. Consequently, Cas9 can bring any fusion protein together with any fusion RNA at any dsDNA sequence by covalent attachment to dCas9 or to sgRNAs, or by noncovalent binding to covalently attached molecules. Knowing that effective concentration is important in regulation of biological processes, CRISPR/Cas9 system can be used as a single unifying factor, capable of mediating biologic interactions. Therefore, it has a great potential for use in investigating and engineering living systems.
For example, transcription is dependent on the assembly of regulatory complexes and their interactions with chromatin. By targeting dCas9 to important binding sites for transcription factors, it should be possible to obstruct the binding of these factors and thereby exclude their role in transcription. Similarly, individual factors with unknown roles could be selectively recruited to almost any desired sequence by dCas9 fusions or sgRNA tethers with only slightly less precision. Together, these capabilities may allow a component-by-component approach to perturbing endogenous gene regulation<ref name="ref7">Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–63 (2013)</ref>.

===Transcriptional activation===

For engineering purposes, it is often useful to directly upregulate the transcription of endogenous genes to a desired level of activity. Experiments with zinc finger effectors and transcription activator–like (TAL) effectors demonstrated that multiple VP64 activator domains localized 5′ of the transcription start site yield synergistic effects. It was shown that Cas9-mediated localization functions similarly with dCas9–VP64. It is important to know that the rate of activation can vary among targeted genes. It requires synergy between multiple Cas9-sgRNA activators for robust transcription. Activation is probably dependent on local chromatin structure, unique interactions with endogenous transcriptional machinery and the Cas9 biochemistry. Elucidation of these effects as well as evaluation of additional Cas9 orthologs will be necessary for fine tuning of control over endogenous transcription.
The capability to upregulate any endogenous gene or combination of genes in trans-acting manner has tremendous implications for ability to investigate and control cellular behavior. In particular, multiplexed sgRNA libraries targeting every known gene could help point out the factors responsible for important cellular processes, such as differentiation.

===Transcriptional repression===

Fusing of repressor domains to zinc finger effector or TAL effector proteins potently suppresses endogenous transcription. By using a similar architecture for dCas9–KRAB or related fusion proteins or sgRNA-based tethers, it should be possible to repress genes with equivalent efficacy and far greater ease of targeting. Indeed, a dCas9–KRAB fusion has been recently shown to induce modest repression using single guide RNAs. Localizing additional repressors and optimizing the structure of the fusion protein could greatly increase the potency of this approach. The ability to repress transcription will not only complement studies using transcriptional activation, but may also be useful for antiviral applications in eukaryotic cells. By preventing the transcription of invading viral genomes, Cas9 repressors could in principle “equip” a transgenic organism with immune to many DNA viruses, targeted with sufficient sgRNAs. This might be a great advantage for crops and domesticated animals.

===Improving specificity===

An increasingly recognized limitation in Cas9-mediated genome engineering applications is their specificity of targeting. The sgRNA-Cas9 complexes are in general tolerant of 1–3 mismatches in their target and occasionally more. It depends on the function of the Cas9 ortholog, the sgRNA architecture, the targeted sequence, the PAM, and also the relative dose and duration of these reagents. Although imperfect Cas9 specificity is a major reason for concern, there are several methods of potentially improving this drawback. Improvements include requiring multiple sgRNA-Cas9 complexes for activity, reducing affinity while increasing cooperativity, establishing competition between inactive and active forms, discovering improved natural orthologs, engineering improved variants and choosing targeting sgRNAs wisely.

===Engineering Cas9-targeted recombinases===

Despite the effectiveness of nuclease-based methods in editing genomes, safe in vivo gene correction in humans remains difficult. Most notably, the introduction of a double-strand break or even a nick at the wrong off-target site can lead to unexpected mutations or rearrangements that may have consequences in carcinogenesis. Site-specific recombinase, and potentially transposase enzymes present fewer problems by tightly controlling generation of DSBs to coordinate donor-target coupling. By fusing the catalytic domain of a small serine recombinase to Cas9, analogous to previous zinc finger and TAL fusions, it may be possible to create an RNA-guided recombinase enzyme. Because the activity of such retargeted fusion recombinases is generally low, extensive directed evolution may be necessary to produce a useful RNA-guided recombinase.

===Discovering or evolving improved Cas9 proteins===

It is possible that certain Cas9 orthologs might prove more specific than the Cas9 from S. pyogenes. It is unlikely that Cas9 proteins with longer PAM requirements will exhibit greater overall specificity, as the selective pressure for accurate recognition of the combined spacer and PAM remains constant. However, Cas9 proteins from species with larger genomes may be somewhat more specific, and those that have undergone frequent horizontal gene transfer along with their CRISPR loci and consequently been selected for avoidance of multiple host genomes are likely the most specific of all.
The best Cas9 proteins identified in nature might be improved by rational design (usually by mutagenesis studies), directed evolution or ideally a combination of the two. One attractive strategy for improving specificity is to reduce the basal Cas9 affinity for DNA, which could be dimminished at target sites by employing two cooperatively binding sgRNAs with complementary 3′ overhangs that target adjacent protospacers. Alternatively, the PAM might be changed to expand the range of targetable sites or enlarged to increase specificity, although such alteration may not be accessible by rational design alone.
PAM alteration and more complex modifications might be accessible using directed evolution, including increasing the overall specificity of each Cas9 monomer. Such experiments must be designed to select for activity at a perfectly matched protospacer. Activity at mismatched sites, preferably those identified as problematic by specificity measurement assays is also important. Ideally, the process would result in selection against many mismatched protospacers at any one time, and the process would be repeated over many rounds of selection. Methods of directed evolution would be convenient for this challenge<ref name="ref7">Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–63 (2013)</ref>..

==Conclusion==

These results show that RNA-programmed genome editing is a straightforward strategy for introducing site-specific genetic changes in human cells. Due to ease of design and effectiveness, in 2013 CRISPR/Cas system took over most the researcher’s attention in field of tools for genome regulation and modification. It seems that it is likely to become competitive with existing approaches based on zinc finger nucleases and transcription activator-like effector nucleases, and could lead to a new generation of experiments in the field of genome engineering for such complex genomes as human<ref name="ref3">Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., Doudna, J., 2013. RNA-programmed genome editing in human cells. Elife 2, e00471. doi:10.7554/eLife.00471.</ref>
Research into the CRISPR-Cas gene editing system continues at great speed. The ease, low cost and speed of designing an RNA guided endonuclease against a DNA target of interest has caught the imagination of worldwide researchers. In beginning of 2014, the crystal structure of ''Streptococcus pyogenes'' Cas9 was published <ref name="ref6">Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–49 (2014)</ref>. This achievement offers the possibility of rational engineering of this RNA-protein complex based on structural information for the first time.
The interest is not in academic sphere; several startups have been created around the technology. Also, reagent companies are already desinging CRISPR reagents for the research community. A few already commercially available products are CRISPR online design tools, CRISPR paired nickases for high specificity genome editing and Cas9 mRNA and expression plasmids.<ref name="ref2">Baker, M., 2014. Gene editing at CRISPR speed. Nat. Biotechnol. 32, 309–12. doi:10.1038/nbt.2863</ref>.

==References==

<references />

[[SB students resources]]

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Luka Smole:

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Luka Smole:

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Luka Smole:

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Luka Smole: /* Introduction */

RNA-guided human genome engineering via Cas9

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Luka Smole:

SB students resources

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Luka Smole: /* List of articles for presentation */

===Introduction to our students resources in Synthetic Biology===
(Marko Dolinar)

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.

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.

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).

===List of articles for presentation===

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):

#[[A synthetic oscillatory network of transcriptional regulators]], Michael B. Elowitz & Stanislas Leibler, Letters to Nature, 2000 - Valter Bergant
#[[Construction of a genetic toggle switch in Escherichia coli]]. Gardner ''et al''., Nature, 2000 - Urban Bezeljak
#Positive feedback in eukaryotic gene networks: cell differentiation by graded to binary response conversion (2001) - Andreja Bratovš
#Chemical synthesis of poliovirus cDNA: Generation of infectious virus in the absence of natural template (2002) - Veronika Jarc
#[[Combinatorial synthesis of genetic networks]]. Guet C.C. ''et al'', Science, 2002 - Maja Remškar
#Engineering a mevalonate pathway in Escherichia coli for production of terpenoids (2003) - Ana Kapraljević
#Programmed population control by cell-cell communication and regulated killing (2004) - Alja Zottel
#Gene regulation at the single-cell level (2005) - Katarina Uršič
#[[A synthetic multicellular system for programmed pattern formation]]. (2005) - Mitja Crček
#Long-term monitoring of bacteria undergoing programmed population control in a microchemostat (2005) - Jana Verbančič
#Tuning genetic control through promoter engineering (2005) - Špela Pohleven
#Production of the antimalarial drug precursor artemisinic acid in engineered yeast (2006) - Živa Marsetič
#An improved zinc-finger nuclease architecture for highly specific genome editing (2007) - Eva Knapič
#Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases (2008) - Tamara Marić
#Synthetic protein scaffolds provide modular control over metabolic flux (2009) - Ana Dolinar
#[[Creation of a bacterial cell controlled by a chemically synthesized genome]]. Gibson, D. G. ''et al.'', Science, 2010 - Eva Lucija Kozak
#A TALE nuclease architecture for efficient genome editing (2011) Jernej Mustar
#Multiplex genome engineering using CRISPR/Cas systems (2013) - Uroš Stupar
#[[RNA-guided human genome engineering via Cas9]]. Mali ''et al''., Science, 2013 - Luka Smole
#[[One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering (2013)]] - Andrej Vrankar

''Please link the title of each paper with your written seminar wiki page. Expand the citation according to the following example:
''
#Emergent bistability by a growth-modulating positive feedback circuit. Tan et al., Nature Chem. Biol., 2009

RNA-guided human genome engineering via Cas9

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Luka Smole:

RNA-guided human genome engineering via Cas9

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Luka Smole: New page: Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., Church, G.M. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/pdf/nihms471334.pdf RNA-guided human...

SB students resources

2015-01-10T17:35:44Z

Luka Smole: /* List of articles for presentation */

===Introduction to our students resources in Synthetic Biology===
(Marko Dolinar)

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.

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.

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).

===List of articles for presentation===

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):

#[[A synthetic oscillatory network of transcriptional regulators]], Michael B. Elowitz & Stanislas Leibler, Letters to Nature, 2000 - Valter Bergant
#[[Construction of a genetic toggle switch in Escherichia coli]]. Gardner ''et al''., Nature, 2000 - Urban Bezeljak
#Positive feedback in eukaryotic gene networks: cell differentiation by graded to binary response conversion (2001) - Andreja Bratovš
#Chemical synthesis of poliovirus cDNA: Generation of infectious virus in the absence of natural template (2002) - Veronika Jarc
#[[Combinatorial synthesis of genetic networks]]. Guet C.C. ''et al'', Science, 2002 - Maja Remškar
#Engineering a mevalonate pathway in Escherichia coli for production of terpenoids (2003) - Ana Kapraljević
#Programmed population control by cell-cell communication and regulated killing (2004) - Alja Zottel
#Gene regulation at the single-cell level (2005) - Katarina Uršič
#[[A synthetic multicellular system for programmed pattern formation]]. (2005) - Mitja Crček
#Long-term monitoring of bacteria undergoing programmed population control in a microchemostat (2005) - Jana Verbančič
#Tuning genetic control through promoter engineering (2005) - Špela Pohleven
#Production of the antimalarial drug precursor artemisinic acid in engineered yeast (2006) - Živa Marsetič
#An improved zinc-finger nuclease architecture for highly specific genome editing (2007) - Eva Knapič
#Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases (2008) - Tamara Marić
#Synthetic protein scaffolds provide modular control over metabolic flux (2009) - Ana Dolinar
#[[Creation of a bacterial cell controlled by a chemically synthesized genome]]. Gibson, D. G. ''et al.'', Science, 2010 - Eva Lucija Kozak
#A TALE nuclease architecture for efficient genome editing (2011) Jernej Mustar
#Multiplex genome engineering using CRISPR/Cas systems (2013) - Uroš Stupar
#[[RNA-guided human genome engineering via Cas9]]. Mali "et al"., Science, 2013 - Luka Smole
#[[One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering (2013)]] - Andrej Vrankar

''Please link the title of each paper with your written seminar wiki page. Expand the citation according to the following example:
''
#Emergent bistability by a growth-modulating positive feedback circuit. Tan et al., Nature Chem. Biol., 2009