Letter | Published:

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

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

Abstract

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.

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Acknowledgements

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

Affiliations

  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

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Contributions

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.

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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.

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DOI

https://doi.org/10.1038/nature24268

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