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DNA targeting specificity of RNA-guided Cas9 nucleases

Nature Biotechnology volume 31, pages 827832 (2013) | Download Citation


The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing1,2,3,4,5,6,7,8,9,10. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

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Change history

  • Corrected online 28 August 2013

    In the version of this article initially published, funding information was left out of the acknowledgments section. The error has been corrected in the HTML and PDF versions of the article.


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We thank A. Shalek, E. Stamenova and D. Gray for expert help with DNA sequencing, R. Barretto for genome-wide PAM analysis, as well as D. Altshuler, P.A. Sharp, and the entire Zhang Lab for their support and advice. P.D.H. is a James Mills Pierce Fellow. D.A.S. is an National Science Foundation pre-doctoral fellow and J.A.W. is supported by a Life Science Research Foundation Fellowship. X.W. is a Howard Hughes Medical Institute International Student Research Fellow and is supported by National Institutes of Health (NIH) grants R01-GM34277 and R01-CA133404 to P.A. Sharp, X.W.'s thesis advisor. G.B. is supported by an NIH Nanomedicine Development Center Award (PN2EY018244).This work is supported by an NIH Director's Pioneer Award (DP1-MH100706), an NIH Transformative R01 grant (R01-DK097768) to D. Altshuler, the Keck, McKnight, Damon Runyon, Searle Scholars, Klingenstein and Simons Foundations, and Bob Metcalfe and Jane Pauley. The authors wish to dedicate this paper to the memory of Officer Sean Collier, for his caring service to the MIT community and for his sacrifice. Reagents are available to the academic community through Addgene, and associated protocols, support forums and computational tools are available through the Zhang lab website (http://www.genome-engineering.org/).

Author information

Author notes

    • Patrick D Hsu
    •  & David A Scott

    These authors contributed equally to this work.


  1. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

    • Patrick D Hsu
    • , David A Scott
    • , Joshua A Weinstein
    • , F Ann Ran
    • , Silvana Konermann
    • , Vineeta Agarwala
    • , Yinqing Li
    • , Ophir Shalem
    •  & Feng Zhang
  2. McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Patrick D Hsu
    • , David A Scott
    • , Joshua A Weinstein
    • , F Ann Ran
    • , Silvana Konermann
    • , Yinqing Li
    • , Ophir Shalem
    •  & Feng Zhang
  3. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Patrick D Hsu
    •  & F Ann Ran
  4. Program in Biophysics, Harvard University, Cambridge, Massachusetts, USA.

    • Vineeta Agarwala
  5. Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, MA 02139, USA.

    • Vineeta Agarwala
  6. Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

    • Eli J Fine
    • , Thomas J Cradick
    •  & Gang Bao
  7. Computational and Systems Biology Graduate Program, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Xuebing Wu
  8. Laboratory of Bacteriology, The Rockefeller University, New York, New York, USA.

    • Luciano A Marraffini


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J.A.W. and F.A.R. contributed equally to this work. P.D.H., D.A.S., F.A.R., S.K. and F.Z. designed and performed the experiments. P.D.H., D.A.S., J.A.W., Y.L., S.K., F.A.R. and F.Z. analyzed the data. V.A. and O.S. contributed computational prediction of CRISPR off-target sites and X.W. performed the northern blot analysis. P.D.H., F.A.R., D.A.S. and F.Z. wrote the manuscript with help from all authors.

Competing interests

A patent application has been filed relating to this work, and the authors plan on making the reagents widely available to the academic community through Addgene and to provide software tools via the Zhang lab website (http://www.genome-engineering.org/).

Corresponding author

Correspondence to Feng Zhang.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Sequences, Supplementary Figures 1–11 and Supplementary Tables 1–4

Excel files

  1. 1.

    Supplementary Table 5

    All sequencing data for Figure 2

  2. 2.

    Supplementary Table 6

    All sequencing data for Figure 3

  3. 3.

    Supplementary Table 7

    All sequencing data for Figure 4

  4. 4.

    Supplementary Table 8

    All sequencing data for expanded set of candidate genomic off-target loci for EMX1 targets 1, 2, 3, and 6

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