Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects

Abstract

CRISPR–Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Identification and characterization of SpCas9 variants bearing substitutions in residues that form non-specific DNA contacts.
Figure 2: Genome-wide specificities of wild-type SpCas9 and SpCas9-HF1 with sgRNAs targeted to standard, non-repetitive sites.
Figure 3: Validation of SpCas9-HF1 specificity improvements by deep sequencing of off-target sites identified by GUIDE-seq.
Figure 4: Genome-wide specificities of wild-type SpCas9 and SpCas9-HF1 with sgRNAs targeted to non-standard, repetitive sites.
Figure 5: Activities of high-fidelity derivatives of SpCas9-HF1 bearing additional substitutions.

Accession codes

Primary accessions

Sequence Read Archive

Data deposits

Plasmids encoding the high-fidelity SpCas9, VQR, and VRQR variants described in this manuscript have been deposited with the non-profit plasmid distribution service Addgene (http://www.addgene.org/crispr-cas). All sequencing data from this study is available through the NCBI Sequence Read Archive (SRA) under accession number SRP066862.

References

  1. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR–Cas9 for genome engineering. Cell 157, 1262–1278 (2014)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. Sander, J. D. & Joung, J. K. CRISPR–Cas systems for editing, regulating and targeting genomes. Nature Biotechnol. 32, 347–355 (2014)

    CAS  Article  Google Scholar 

  3. Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR–Cas9. Science 346, 1258096 (2014)

    PubMed  Article  CAS  Google Scholar 

  4. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR–Cas nucleases in human cells. Nature Biotechnol. 31, 822–826 (2013)

    CAS  Article  Google Scholar 

  5. Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnol. 31, 827–832 (2013)

    CAS  Article  Google Scholar 

  6. Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnol. 31, 839–843 (2013)

    CAS  Article  Google Scholar 

  7. Cradick, T. J., Fine, E. J., Antico, C. J. & Bao, G. CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 41, 9584–9592 (2013)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nature Biotechnol. 33, 187–197 (2015)

    CAS  Article  Google Scholar 

  9. Frock, R. L. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nature Biotechnol. 33, 179–186 (2015)

    CAS  Article  Google Scholar 

  10. Wang, X. et al. Unbiased detection of off-target cleavage by CRISPR–Cas9 and TALENs using integrase-defective lentiviral vectors. Nature Biotechnol. 33, 175–178 (2015)

    CAS  Article  Google Scholar 

  11. Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR–Cas9 off-target effects in human cells. Nature Methods 12, 237–243 (2015)

    CAS  PubMed  Article  Google Scholar 

  12. Lin, Y. et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 42, 7473–7485 (2014)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Cho, S. W. et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24, 132–141 (2014)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR–Cas nuclease specificity using truncated guide RNAs. Nature Biotechnol. 32, 279–284 (2014)

    CAS  Article  Google Scholar 

  15. Kleinstiver, B. P. et al. Engineered CRISPR–Cas9 nucleases with altered specificities. Nature 523, 481–485 (2015)

    ADS  PubMed  PubMed Central  Article  CAS  Google Scholar 

  16. Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnol. 31, 833–838 (2013)

    CAS  Article  Google Scholar 

  17. Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Tsai, S. Q. et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nature Biotechnol. 32, 569–576 (2014)

    CAS  Article  Google Scholar 

  19. Guilinger, J. P., Thompson, D. B. & Liu, D. R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nature Biotechnol. 32, 577–582 (2014)

    CAS  Article  Google Scholar 

  20. Wyvekens, N., Topkar, V. V., Khayter, C., Joung, J. K. & Tsai, S. Q. Dimeric CRISPR RNA-guided FokI-dCas9 nucleases directed by truncated gRNAs for highly specific genome editing. Hum. Gene Ther. 26, 425–431 (2015)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nature Biotechnol. 31, 233–239 (2013)

    CAS  Article  Google Scholar 

  24. Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. Jinek, M. et al. RNA-programmed genome editing in human cells. Elife 2, e00471 (2013)

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  26. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. Anders, C., Niewoehner, O., Duerst, A. & Jinek, M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569–573 (2014)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nature Biotechnol. 30, 460–465 (2012)

    CAS  Article  Google Scholar 

  31. Jiang, F., Zhou, K., Ma, L., Gressel, S. & Doudna, J. A. A Cas9-guide RNA complex preorganized for target DNA recognition. Science 348, 1477–1481 (2015)

    ADS  CAS  PubMed  Article  Google Scholar 

  32. Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, http://dx.doi.org/10.1126/science.1247997 (2014)

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  33. Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Guilinger, J. P. et al. Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. Nature Methods 11, 429–435 (2014)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. Sternberg, S. H., LaFrance, B., Kaplan, M. & Doudna, J. A. Conformational control of DNA target cleavage by CRISPR–Cas9. Nature 527, 110–113 (2015)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. Knight, S. C. et al. Dynamics of CRISPR–Cas9 genome interrogation in living cells. Science 350, 823–826 (2015)

    ADS  CAS  PubMed  Article  Google Scholar 

  37. Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science http://dx.doi.org/10.1126/science.aad5227 (2015)

  38. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Esvelt, K. M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nature Methods 10, 1116–1121 (2013)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Hou, Z. et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc. Natl Acad. Sci. USA 110, 15644–15649 (2013)

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  41. Fonfara, I. et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR–Cas systems. Nucleic Acids Res. 42, 2577–2590 (2014)

    CAS  PubMed  Article  Google Scholar 

  42. Kleinstiver, B. P. et al. Broadening the targeting range of Staphylococcus aureus CRISPR–Cas9 by modifying PAM recognition. Nature Biotechnol. 33, 1293–1298 (2015)

    CAS  Article  Google Scholar 

  43. Zetsche, B. et al. Cpf1 is a single RNA-Guided endonuclease of a class 2 CRISPR–Cas system. Cell 163, 759–771 (2015)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. Shmakov, S. et al. Discovery and functional characterization of diverse class 2 CRISPR–Cas systems. Mol. Cell 60, 385–397 (2015)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. Kearse, M. et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647–1649 (2012)

    PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

B.P.K. is supported by a Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowship. V.P. was supported by the Massachusetts General Hospital (MGH) Department of Pathology. S.Q.T. is supported by an MGH Tosteson and Fund for Medical Discovery Fellowship. J.K.J. is supported by a US National Institutes of Health (NIH) Director’s Pioneer Award (DP1 GM105378), NIH R01 GM107427, NIH R01 GM088040, and the Jim and Ann Orr MGH Research Scholar Award.

Author information

Authors and Affiliations

Authors

Contributions

B.P.K., V.P., and J.K.J. conceived of and designed experiments. B.P.K., V.P., and M.S.P. performed all experiments. N.T.N. contributed to GUIDE-seq library preparation. B.P.K., V.P., M.S.P., S.Q.T., and Z.Z. analysed the data. B.P.K., V.P., and J.K.J. wrote the manuscript with input from all the authors.

Corresponding author

Correspondence to J. Keith Joung.

Ethics declarations

Competing interests

J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Editas Medicine, Hera Testing Laboratories, Poseida Therapeutics, and Transposagen Biopharmaceuticals. J.K.J.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. A patent application has been filed for high-fidelity Cas9 variants.

Extended data figures and tables

Extended Data Figure 1 SpCas9 interaction with the sgRNA and target DNA.

a, Schematic illustrating the SpCas9–sgRNA complex, with base pairing between the sgRNA and target DNA. b, Structural representation of the SpCas9-sgRNA complex bound to the target DNA, from PDB accession code 4UN3 (ref. 29). The four residues that form hydrogen bond contacts to the target-strand DNA backbone are highlighted in blue, the HNH domain is hidden for visualization purposes.

Extended Data Figure 2 On-target activities of high-fidelity SpCas9 variants.

a, b, EGFP disruption activities of wild-type SpCas9 and SpCas9-HF1 (a) and SpCas9-HF1-derivative variants (b) in human cells. SpCas9-HF1 contains N497A, R661A, Q695A, and Q926A substitutions; HF2 = HF1 + D1135E; HF3 = HF1 + L169A; HF4 = HF1 + Y450A. Error bars represent s.e.m. for n = 3; mean level of background EGFP loss represented by the red dashed line.

Extended Data Figure 3 On-target activity comparisons of wild-type and SpCas9-HF1 with various sgRNAs used for GUIDE-seq experiments.

a, c, Mean GUIDE-seq tag integration at the intended on-target site for GUIDE-seq experiments shown in Figs 2a and Extended Data Fig. 5 (a and c, respectively), quantified by restriction-fragment length polymorphism assay. Error bars represent s.e.m. for n = 3. b, d, Mean percent modification at the intended on-target site for GUIDE-seq experiments shown in Fig. 2a and Extended Data Fig. 5 (b and d, respectively), detected by T7 endonuclease I assay. Error bars represent s.e.m. for n = 3.

Extended Data Figure 4 Positional summary of off-target sites identified by GUIDE-seq.

a, b, Heat maps derived from GUIDE-seq data with sgRNAs targeting non-repetitive (a), or repetitive or homopolymeric sites (b) in the genome are shown. Base frequencies in the set of all potential genomic off-target sites (weighted equally) with NGG PAMs and five or fewer mutations for each sgRNA are shown on the left. Summaries of off-target sites identified by GUIDE-seq for wild-type SpCas9 and SpCas9-HF1 (both weighted by read count) are shown on the right. Yellow box outlines denote on-target bases at each position. Positions (20–1) are shown below the heat maps, with 1 being the most PAM-proximal position. Note the presence of mismatches that would be expected to create potential wobble interactions (G→A or T→C) at certain positions among the off-target sites induced by wild-type SpCas9 and that SpCas9-HF1 appears to reduce off-target activity without any obvious positional bias.

Extended Data Figure 5 Genome-wide cleavage specificity of wild-type SpCas9 and SpCas9-HF1 with sgRNAs targeted to non-standard, repetitive sites.

a, GUIDE-seq profiles of wild-type SpCas9 and SpCas9-HF1 using two sgRNAs known to cleave large numbers of off-target sites4,8. GUIDE-seq read counts represent a measure of cleavage efficiency at a given site. Mismatched positions within the spacer or PAM are highlighted in colour red circles indicate off-target sites likely to have the indicated bulge12 at the sgRNA–DNA interface, blue circles indicate sites that may have an alternative gapped alignment relative to the one shown (see Extended Data Fig. 6). Off-target sites marked with red circles are not included in the counts of Fig. 4b, sites marked with blue circles are counted with the number of mismatches in the non-gapped alignment for Fig. 4b.

Extended Data Figure 6 Potential alternate alignments for VEGFA site 2 off-target sites.

Ten VEGFA site 2 off-target sites identified by GUIDE-seq (left) that may potentially be recognized as off-target sites with single nucleotide gaps12 (right), aligned using Geneious45 version 8.1.6 (http://www.geneious.com).

Extended Data Figure 7 Activities of wild-type SpCas9 and SpCas9-HF1 with truncated or 5′ mismatched sgRNAs14.

a, EGFP disruption activities of wild-type SpCas9 and SpCas9-HF1 using full-length or truncated sgRNAs. b, EGFP disruption activities of wild-type SpCas9 and SpCas9-HF1 using sgRNAs that encode a matched 5′ non-G nucleotide or an intentionally mismatched 5′ G nucleotide. For both panels, error bars represent s.e.m. for n = 3, and the mean level of background EGFP loss observed in control experiments is represented by the red dashed line.

Extended Data Figure 8 Altering the PAM recognition specificity of SpCas9-HF1.

a, Comparison of the mean per cent modification of on-target endogenous human sites by the SpCas9-VQR variant (ref. 15) and an improved SpCas9-VRQR variant using 8 sgRNAs, quantified by T7 endonuclease I assay. Both variants are engineered to recognize an NGAN PAM. Error bars represent s.e.m. for n = 3. b, On-target EGFP disruption activities of SpCas9-VQR and SpCas9-VRQR compared to their -HF1 counterparts using eight sgRNAs. Error bars represent s.e.m. for n = 3; mean level of background EGFP loss in negative controls represented by the red dashed line. c, Comparison of the mean on-target per cent modification by SpCas9-VQR and SpCas9-VRQR compared to their -HF1 variants at eight endogenous human gene sites, quantified by T7 endonuclease I assay. Error bars represent s.e.m. for n = 3; ND, not detectable. d, Summary of the fold-change in on-target activity when using SpCas9-VQR or SpCas9-VRQR compared to their corresponding -HF1 variants (from b and c). The median and interquartile range are shown, the interval showing greater than 70% of wild-type activity is highlighted in green.

Extended Data Figure 9 Titrations of wild-type SpCas9 and SpCas9-HF1 expression plasmid amounts.

Human cell EGFP disruption activities from transfections with varying amounts of wild-type and SpCas9-HF1 expression plasmids. For all transfections, the amount of sgRNA-containing plasmid was fixed at 250 ng. Two sgRNAs targeting different sites were used; Error bars represent s.e.m. for n = 3; mean level of background EGFP loss in negative controls is represented by the red dashed line.

Extended Data Table 1 Summary of potential mismatched sites in the reference human genome for the ten sgRNAs examined by GUIDE-seq

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, additional references and Supplementary Sequences (a subset of plasmids used in this study). (PDF 297 kb)

Supplementary Table 1

This table contains the sgRNA targets. (XLSX 17 kb)

Supplementary Table 2

This table contains the oligonucleotides used in this study. (XLSX 11 kb)

Supplementary Table 3

This table, which has multiply tabs, contains the p-values for data from Figures 1 and 5. (XLSX 66 kb)

Supplementary Table 4

This table, which has multiply tabs, contains the summary of GUIDE-seq data. (XLSX 112 kb)

Supplementary Table 5

This table, which has multiply tabs, contains the targeted deep sequencing amplicons and data. (XLSX 82 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kleinstiver, B., Pattanayak, V., Prew, M. et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016). https://doi.org/10.1038/nature16526

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature16526

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing