RNA-guided human genome engineering via Cas9 - Revision history

Luka Smole at 08:10, 13 January 2015

2015-01-13T08:10:44Z

← Older revision		Revision as of 08:10, 13 January 2015
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	==Introduction==		==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.		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.

Luka Smole at 17:49, 12 January 2015

2015-01-12T17:49:29Z

Luka Smole at 17:47, 12 January 2015

2015-01-12T17:47:04Z

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 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.
 ==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 ''Streptococcus pyogenes'' Cas9 was published 6. This achievement offers the possibility of rational engineering of the this RNA-protein complex based on structural information for the first time.
+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>.

Luka Smole: /* Conclusion */

2015-01-11T15:29:30Z

Conclusion

← Older revision		Revision as of 15:29, 11 January 2015
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	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>		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 ''Streptococcus pyogenes'' Cas9 was published 6. This achievement offers the possibility of rational engineering of the this RNA-protein complex based on structural information for the first time.		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 ''Streptococcus pyogenes'' Cas9 was published 6. This achievement offers the possibility of rational engineering of the 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 ~~rushing to create~~ CRISPR reagents for the research community<ref name="ref2">Baker, M., 2014. Gene editing at CRISPR speed. Nat. Biotechnol. 32, 309–12. doi:10.1038/nbt.2863</ref>.		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==

Luka Smole at 15:22, 11 January 2015

2015-01-11T15:22:19Z

← Older revision		Revision as of 15:22, 11 January 2015
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	===Modifying the native AAVS1 locus===		===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.		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.


	==Conclusion==		==Conclusion==

Luka Smole at 14:45, 11 January 2015

2015-01-11T14:45:04Z

← Older revision		Revision as of 14:45, 11 January 2015
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	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.		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.		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.		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.

Luka Smole at 09:45, 11 January 2015

2015-01-11T09:45:40Z

← Older revision		Revision as of 09:45, 11 January 2015
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	*Note:		*Note:
	While writing this seminar, I put a lot of effort in summarizing this research in most comprehensible manner ~~as possible~~. 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).		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==		==CRISPR/Cas9 system==

Luka Smole: /* Introduction */

2015-01-10T18:26:57Z

Introduction

← Older revision		Revision as of 18:26, 10 January 2015
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	*Note:		*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).		While writing this seminar, I put a lot of effort in summarizing this research in most comprehensible manner as possible. 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==		==CRISPR/Cas9 system==

Luka Smole at 18:26, 10 January 2015

2015-01-10T18:26:23Z

← Older revision		Revision as of 18:26, 10 January 2015
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	Luka Smole		Luka Smole


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


	==Introduction==		==Introduction==

Luka Smole at 18:23, 10 January 2015

2015-01-10T18:23:32Z

← Older revision		Revision as of 18:23, 10 January 2015
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	==Introduction==		==Introduction==

	In this seminar I will write about CRISPR/Cas system, in this case constructed for human genome engineering. In this research 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.		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.

← Older revision		Revision as of 17:49, 12 January 2015
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	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>.		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==		==Design of CRISPR/Cas9 system for RNA-guided human genome engineering==
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	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.		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.		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===		===Targeting in native endogenous AAVS1 locus===
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	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.		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.		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===		===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%.		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===		===Bioinformatically generated gRNA-targetable sequences===
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	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.		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>..		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==		==Conclusion==