Article | Published:

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

Nature volume 529, pages 490495 (28 January 2016) | Download Citation

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.

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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. 1.

    , & Development and applications of CRISPR–Cas9 for genome engineering. Cell 157, 1262–1278 (2014)

  2. 2.

    & CRISPR–Cas systems for editing, regulating and targeting genomes. Nature Biotechnol. 32, 347–355 (2014)

  3. 3.

    & Genome editing. The new frontier of genome engineering with CRISPR–Cas9. Science 346, 1258096 (2014)

  4. 4.

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

  5. 5.

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

  6. 6.

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

  7. 7.

    , , & CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 41, 9584–9592 (2013)

  8. 8.

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

  9. 9.

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

  10. 10.

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

  11. 11.

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

  12. 12.

    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)

  13. 13.

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

  14. 14.

    , , , & Improving CRISPR–Cas nuclease specificity using truncated guide RNAs. Nature Biotechnol. 32, 279–284 (2014)

  15. 15.

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

  16. 16.

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

  17. 17.

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

  18. 18.

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

  19. 19.

    , & Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nature Biotechnol. 32, 577–582 (2014)

  20. 20.

    , , , & Dimeric CRISPR RNA-guided FokI-dCas9 nucleases directed by truncated gRNAs for highly specific genome editing. Hum. Gene Ther. 26, 425–431 (2015)

  21. 21.

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

  22. 22.

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

  23. 23.

    , , , & RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nature Biotechnol. 31, 233–239 (2013)

  24. 24.

    , , , & DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014)

  25. 25.

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

  26. 26.

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

  27. 27.

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

  28. 28.

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

  29. 29.

    , , & Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569–573 (2014)

  30. 30.

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

  31. 31.

    , , , & A Cas9-guide RNA complex preorganized for target DNA recognition. Science 348, 1477–1481 (2015)

  32. 32.

    et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, (2014)

  33. 33.

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

  34. 34.

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

  35. 35.

    , , & Conformational control of DNA target cleavage by CRISPR–Cas9. Nature 527, 110–113 (2015)

  36. 36.

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

  37. 37.

    et al. Rationally engineered Cas9 nucleases with improved specificity. Science (2015)

  38. 38.

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

  39. 39.

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

  40. 40.

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

  41. 41.

    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)

  42. 42.

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

  43. 43.

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

  44. 44.

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

  45. 45.

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

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

Author notes

    • Benjamin P. Kleinstiver
    •  & Vikram Pattanayak

    These authors contributed equally to this work.

Affiliations

  1. Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA

    • Benjamin P. Kleinstiver
    • , Vikram Pattanayak
    • , Michelle S. Prew
    • , Shengdar Q. Tsai
    • , Nhu T. Nguyen
    •  & J. Keith Joung
  2. Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Benjamin P. Kleinstiver
    • , Vikram Pattanayak
    • , Shengdar Q. Tsai
    •  & J. Keith Joung
  3. Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China

    • Zongli Zheng

Authors

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  2. Search for Vikram Pattanayak in:

  3. Search for Michelle S. Prew in:

  4. Search for Shengdar Q. Tsai in:

  5. Search for Nhu T. Nguyen in:

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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.

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.

Corresponding author

Correspondence to J. Keith Joung.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

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

Excel files

  1. 1.

    Supplementary Table 1

    This table contains the sgRNA targets.

  2. 2.

    Supplementary Table 2

    This table contains the oligonucleotides used in this study.

  3. 3.

    Supplementary Table 3

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

  4. 4.

    Supplementary Table 4

    This table, which has multiply tabs, contains the summary of GUIDE-seq data.

  5. 5.

    Supplementary Table 5

    This table, which has multiply tabs, contains the targeted deep sequencing amplicons and data.

About this article

Publication history

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DOI

https://doi.org/10.1038/nature16526

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