Letter | Published:

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

Nature volume 550, pages 407410 (19 October 2017) | Download Citation


The RNA-guided CRISPR–Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1,2,3,4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown5,6,7,8,9. Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we design a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions

Sequence Read Archive


  1. 1.

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

  2. 2.

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

  3. 3.

    , & Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013)

  4. 4.

    & A decade of discovery: CRISPR functions and applications. Nat. Microbiol. 2, 17092 (2017)

  5. 5.

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

  6. 6.

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

  7. 7.

    & Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat. Rev. Genet. 17, 300–312 (2016)

  8. 8.

    et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016)

  9. 9.

    et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016)

  10. 10.

    , , , & A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9. Sci. Adv. 3, eaao0027 (2017)

  11. 11.

    , & Lessons from enzyme kinetics reveal specificity principles for RNA-guided nucleases in RNA interference and CRISPR-based genome editing. Cell Syst. 4, 21–29 (2017)

  12. 12.

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

  13. 13.

    et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science 351, 867–871 (2016)

  14. 14.

    , , , & Striking plasticity of CRISPR-Cas9 and key role of non-target DNA, as revealed by molecular simulations. ACS Cent. Sci. 2, 756–763 (2016)

  15. 15.

    , , , & CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations. Proc. Natl Acad. Sci. USA 114, 7260–7265 (2017)

  16. 16.

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

  17. 17.

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

  18. 18.

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

  19. 19.

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

  20. 20.

    , , & Measurements of internal distance changes of the 30S ribosome using FRET with multiple donor-acceptor pairs: quantitative spectroscopic methods. J. Mol. Biol. 351, 1123–1145 (2005)

  21. 21.

    et al. Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes. Proc. Natl Acad. Sci. USA 111, 9798–9803 (2014)

  22. 22.

    et al. Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage. PLoS ONE 9, e109213 (2014)

  23. 23.

    et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1247997 (2014)

  24. 24.

    et al. Rational design of a split-Cas9 enzyme complex. Proc. Natl Acad. Sci. USA 112, 2984–2989 (2015)

  25. 25.

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

  26. 26.

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

  27. 27.

    , , & Open-source guideseq software for analysis of GUIDE-seq data. Nat. Biotechnol. 34, 483 (2016)

  28. 28.

    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)

  29. 29.

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

Download references


We thank A. V. Wright, S. N. Floor, J. C. Cofsky, D. Burstein, C. Fellman, B. L. Oakes and O. Mavrothalassitis for discussions and reading the manuscript, M. S. Prew for technical assistance, and J. M. Lopez for assistance with GUIDE-seq data processing. J.S.C. and L.B.H. are supported by National Science Foundation Graduate Research Fellowships, and B.P.K. from Banting (Natural Sciences and Engineering Research Council of Canada) and Charles A. King Trust Postdoctoral Fellowships. J.A.D. is an Investigator of the Howard Hughes Medical Institute. This work was supported by the National Institutes of Health (GM094522 and GM118773 (A.Y.), R35 GM118158 (J.K.J.)), National Science Foundation (MCB-1617028 (A.Y.) and MCB-1244557 (J.A.D.)), and the Desmond and Ann Heathwood MGH Research Scholar Award (J.K.J.).

Author information

Author notes

    • Janice S. Chen
    • , Yavuz S. Dagdas
    •  & Benjamin P. Kleinstiver

    These authors contributed equally to this work.

    • Samuel H. Sternberg

    Present address: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA


  1. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA

    • Janice S. Chen
    • , Lucas B. Harrington
    • , Ahmet Yildiz
    •  & Jennifer A. Doudna
  2. Biophysics Graduate Group, University of California, Berkeley, California 94720, USA

    • Yavuz S. Dagdas
  3. Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA

    • Benjamin P. Kleinstiver
    • , Moira M. Welch
    • , Alexander A. Sousa
    •  & J. Keith Joung
  4. Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA

    • Benjamin P. Kleinstiver
    • , Moira M. Welch
    • , Alexander A. Sousa
    •  & J. Keith Joung
  5. Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Benjamin P. Kleinstiver
    •  & J. Keith Joung
  6. Department of Chemistry, University of California, Berkeley, California 94720, USA

    • Samuel H. Sternberg
    •  & Jennifer A. Doudna
  7. Department of Physics, University of California, Berkeley, California 94720, USA

    • Ahmet Yildiz
  8. Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA

    • Jennifer A. Doudna
  9. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Jennifer A. Doudna


  1. Search for Janice S. Chen in:

  2. Search for Yavuz S. Dagdas in:

  3. Search for Benjamin P. Kleinstiver in:

  4. Search for Moira M. Welch in:

  5. Search for Alexander A. Sousa in:

  6. Search for Lucas B. Harrington in:

  7. Search for Samuel H. Sternberg in:

  8. Search for J. Keith Joung in:

  9. Search for Ahmet Yildiz in:

  10. Search for Jennifer A. Doudna in:


J.S.C., Y.S.D. and B.P.K. contributed equally to the work, and conceived and designed experiments with input from L.B.H., S.H.S., J.K.J., A.Y. and J.A.D. J.S.C. performed protein expression, labelling and biochemical experiments. Y.S.D. performed single-molecule fluorescence assays and related data analysis. B.P.K. and M.M.W. performed human cell-based assays, and B.P.K. and A.A.S. performed and analysed GUIDE-seq experiments. J.S.C., Y.S.D., B.P.K., J.K.J., A.Y. and J.A.D. wrote the manuscript.

Competing interests

J.K.J. has financial interests in Beacon Genomics, Beam Therapeutics, Editas Medicine, Pairwise Plants, 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. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, and Intellia Therapeutics; a scientific adviser to Caribou, Intellia, eFFECTOR Therapeutics and Driver; and executive director of the Innovative Genomics Institute at the University of California, Berkeley and University of California, San Francisco. S.H.S. is an employee of Caribou Biosciences, Inc. S.H.S., J.S.C., and J.A.D. are inventors on a patent application entitled ‘Reporter Cas9 variants and methods of use thereof’ (PCT/US2016/036754), filed by The Regents of the University of California. B.P.K. and J.K.J. are inventors on a patent application entitled ‘Engineered CRISPR-Cas9 nucleases’ (US 15/060,424), filed by The General Hospital Corporation. J.S.C., Y.S.D., B.P.K., A.Y., J.K.J., and J.A.D. have filed a patent application related to this work through The General Hospital Corporation and The Regents of the University of California.

Corresponding author

Correspondence to Jennifer A. Doudna.

Reviewer Information Nature thanks A. Ke and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figure

    This file contains the uncropped gel images from polyacrylamide gel electrophoresis experiments presented in the manuscript.

  2. 2.

    Reporting Summary

Excel files

  1. 1.

    Supplementary Table 1

    This file contains GUIDE-seq data.

  2. 2.

    Supplementary Table 2

    This file contains DNA plasmids and proteins used in this study. All enhanced specificity, high-fidelity, cluster and hyper-accurate SpCas9 variants tested in this study, with Addgene ID numbers for deposited plasmids. The HNH, REC2 or REC3 subscript designation with an enhanced specificity, high-fidelity or cluster SpCas9 variant denotes combination of residue substitutions with indicated FRET construct.

  3. 3.

    Supplementary Table 3

    This file contains a list of nucleic acids used in the study.

About this article

Publication history






Further reading


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.