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


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 ( All sequencing data from this study is available through the NCBI Sequence Read Archive (SRA) under accession number SRP066862.


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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.


  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


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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.

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