Abstract

RNA-guided CRISPR–Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9–sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.

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Acknowledgements

The authors thank R. Haurwitz, S. Sternberg, B. McClung, and the members of Caribou Biosciences for providing helpful comments on the manuscript.

Author information

Author notes

    • Brittnee N Jones
    • , Mark Cigan
    •  & Andrew P May

    Present addresses: Omicia, Inc., Oakland, California, USA (B.N.J.); Genus Research, DeForest, Wisconsin, USA (M.C.) and Chan Zuckerberg Biohub, San Francisco, California, USA (A.P.M.).

    • Peter Cameron
    •  & Chris K Fuller

    These authors contributed equally to this work.

Affiliations

  1. Caribou Biosciences, Berkeley, California, USA.

    • Peter Cameron
    • , Chris K Fuller
    • , Paul D Donohoue
    • , Brittnee N Jones
    • , Matthew S Thompson
    • , Matthew M Carter
    • , Scott Gradia
    • , Bastien Vidal
    • , Elizabeth Garner
    • , Euan M Slorach
    • , Elaine Lau
    • , Lynda M Banh
    • , Alexandra M Lied
    • , Leslie S Edwards
    • , Alexander H Settle
    • , Daniel Capurso
    •  & Andrew P May
  2. DuPont Pioneer, Johnston, Iowa, USA.

    • Victor Llaca
    • , Stéphane Deschamps
    • , Mark Cigan
    •  & Joshua K Young

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Contributions

J.K.Y., V.L., S.D., M.C., A.P.M. and P.D.D. designed critical proof-of-concept experiments to initially develop the SITE-Seq protocol. P.C. performed all SITE-Seq experiments in this study; B.N.J. performed amplicon digestion experiments; M.S.T. and L.M.B. performed sequencing for cell-based validation experiments. P.C., E.M.S., B.V., E.G. and E.L. performed cell-based validation experiments. S.G., A.M.L. and L.S.E. generated sgRNA and other reagents. P.C., C.K.F., P.D.D., M.M.C., D.C., M.C., B.N.J., A.H.S., J.K.Y. and A.P.M. analyzed the data. P.C., C.K.F., P.D.D., M.C., J.K.Y. and A.P.M. wrote the manuscript.

Competing interests

A.P.M., P.D.D., P.C., C.K.F., M.S.T., M.M.C., S.G., B.N.J., E.L., E.M.S., B.V., E.L., L.M.B., A.M.L., L.S.E., A.H.S., and D.C. are current or former employees of Caribou Biosciences, Inc., a company that develops and commercializes genome engineering technologies; and such individuals may own shares or stock options in Caribou Biosciences, Inc. J.K.Y., V.L., and S.D. are employees of DuPont Pioneer. Patent applications have been filed describing this methodology; see, for example, PCT publication no. WO2014/164466, published October 9, 2014.

Corresponding authors

Correspondence to Joshua K Young or Andrew P May.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–4

  2. 2.

    Supplementary Protocol

    SITE-Seq Supplementary Protocol

Excel files

  1. 1.

    Supplementary Table 1–9

    SITE-Seq target sites recovered

  2. 2.

    Supplementary Table 10

    SITE-Seq target sites recovered with VEGFA sgRNP that were segregated by MEME into a second motif.

  3. 3.

    Supplementary Table 11

    Biochemical cleavage of amplicons containing SITE-Seq target sites, as a function of sgRNP concentration.

  4. 4.

    Supplementary Table 12–19

    SITE-Seq target sites examined in cell-based validation.

  5. 5.

    Supplementary Table 20–22

    SITE-Seq target sites examined in cell-based validation with a panel of delivery methods.

  6. 6.

    Supplementary Table 23

    SITE-Seq with high sequencing coverage.

  7. 7.

    Supplementary Table 24

    Comparing SITE-Seq data with in silico approaches

  8. 8.

    Supplementary Table 25-26

    Oligonucleotides used in SITE-Seq.

Text files

  1. 1.

    Supplementary Software

    SITE-Seq feature calling function

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DOI

https://doi.org/10.1038/nmeth.4284