Rationally engineered Cas9 nucleases with improved specificity

doi:10.1126/science.aad5227

. 2016 Jan 1;351(6268):84-8.

doi: 10.1126/science.aad5227. Epub 2015 Dec 1.

Rationally engineered Cas9 nucleases with improved specificity

Ian M Slaymaker¹, Linyi Gao², Bernd Zetsche¹, David A Scott¹, Winston X Yan³, Feng Zhang⁴

Affiliations

¹ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
² Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Graduate Program in Biophysics, Harvard Medical School, Boston, MA 02115, USA. Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, MA 02115, USA.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. zhang@broadinstitute.org.

PMID: 26628643
PMCID: PMC4714946
DOI: 10.1126/science.aad5227

Rationally engineered Cas9 nucleases with improved specificity

Ian M Slaymaker et al. Science. 2016.

. 2016 Jan 1;351(6268):84-8.

doi: 10.1126/science.aad5227. Epub 2015 Dec 1.

Authors

Ian M Slaymaker¹, Linyi Gao², Bernd Zetsche¹, David A Scott¹, Winston X Yan³, Feng Zhang⁴

Affiliations

¹ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
² Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Graduate Program in Biophysics, Harvard Medical School, Boston, MA 02115, USA. Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, MA 02115, USA.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. zhang@broadinstitute.org.

PMID: 26628643
PMCID: PMC4714946
DOI: 10.1126/science.aad5227

Abstract

The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that "enhanced specificity" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.

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Figures

**Fig. 1. Structure-guided mutagenesis improves specificity of SpCas9**
(A) A model of Cas9 unwinding highlighting locations of charge on DNA and the nt-groove. The nt-groove between the RuvC (teal) and HNH (magenta) domains stabilize DNA unwinding through non-specific DNA interactions with the non-complementary strand. RNA:cDNA and Cas9:ncDNA interactions drive DNA unwinding (top arrow) in competition against cDNA:ncDNA rehybridization (bottom arrow). (B) A crystal structure of SpCas9 (PDB ID 4UN3) showing the nt-groove situated between the HNH (magenta) and RuvC (teal) domains. The non-target DNA strand (red) was manually modeled into the nt-groove (inset). (C) Screen of alanine single mutants for improvement in specificity. The top five specificity conferring mutants are highlighted in red. (D) Assessment of top single mutants at additional off-target loci. (E) Combination mutants improve specificity compared to single mutants. eSpCas9(1.0) and eSpCas9(1.1) are highlighted in red.

**Fig. 2. SpCas9 mutants maintain on-target efficiency**
(A) Assessment of mutants for efficient on-target cutting with 24 sgRNAs targeted to 10 genomic loci. (B) Tukey plot of normalized on-target indel formation for mutants. (C) Western blot of SpCas9 using anti-SpCas9 antibody.

**Fig. 3. SpCas9 mutants exhibited increased sensitivity to single and double base mismatches between the guide RNA and target DNA**
(A) Schematic showing design of mismatched guide sequences against *VEGFA*(1). (B) Heat maps showing indel % of guide sequences with a single base mismatch. (C) Indel formation with guide sequences containing consecutive transversion mismatches.

**Fig. 4. Unbiased genome-wide off-target profile of mutants using BLESS**
(A and B) Manhattan plots of genome-wide DSB clusters generated by each SpCas9 mutant using the *EMX1*(1) and *VEGFA*(1) targeting guides. (C and D) Targeted deep sequencing validation of off-target sites identified in BLESS. Off-target sites are ordered by DSB score (blue heatmap). Green heatmaps indicates sequence similarity between target and off-target sequences.

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References

1. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–823. doi: 10.1126/science.1231143. - DOI - PMC - PubMed
1. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–826. doi: 10.1126/science.1232033. - DOI - PMC - PubMed
1. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31:827–832. doi: 10.1038/nbt.2647. - DOI - PMC - PubMed
1. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013;31:822–826. doi: 10.1038/nbt.2623. - DOI - PMC - PubMed
1. Zetsche B, Volz SE, Zhang F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat Biotechnol. 2015;33:139–142. doi: 10.1038/nbt.3149. - DOI - PMC - PubMed

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[1] Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–823. doi: 10.1126/science.1231143. - DOI - PMC - PubMed

[2] Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–823. doi: 10.1126/science.1231143. - DOI - PMC - PubMed

[3] Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–826. doi: 10.1126/science.1232033. - DOI - PMC - PubMed

[4] Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–826. doi: 10.1126/science.1232033. - DOI - PMC - PubMed

[5] Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31:827–832. doi: 10.1038/nbt.2647. - DOI - PMC - PubMed

[6] Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31:827–832. doi: 10.1038/nbt.2647. - DOI - PMC - PubMed

[7] Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013;31:822–826. doi: 10.1038/nbt.2623. - DOI - PMC - PubMed

[8] Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013;31:822–826. doi: 10.1038/nbt.2623. - DOI - PMC - PubMed

[9] Zetsche B, Volz SE, Zhang F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat Biotechnol. 2015;33:139–142. doi: 10.1038/nbt.3149. - DOI - PMC - PubMed

[10] Zetsche B, Volz SE, Zhang F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat Biotechnol. 2015;33:139–142. doi: 10.1038/nbt.3149. - DOI - PMC - PubMed

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Rationally engineered Cas9 nucleases with improved specificity

Affiliations

Rationally engineered Cas9 nucleases with improved specificity

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