Article | Published:

Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells

Nature Methods volume 12, pages 237243 (2015) | Download Citation

Abstract

Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5′ ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.

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Acknowledgements

This research was supported by grants from the Institute for Basic Science (IBS-R021-D1) to J.-S.K., Korea Health Industry Development Institute (HI14C1277) to J.-I.K., and Korea Institute of Planning and Evaluation for Technology of Food, Agriculture, Forestry and Fisheries (311011-05-3-SB010) to S.K.

Author information

Author notes

    • Sangsu Bae

    Present address: Department of Chemistry, Hanyang University, Seoul, South Korea.

    • Daesik Kim
    •  & Sangsu Bae

    These authors contributed equally to this work.

Affiliations

  1. Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea.

    • Daesik Kim
    • , Sangsu Bae
    •  & Jin-Soo Kim
  2. Department of Chemistry, Seoul National University, Seoul, South Korea.

    • Daesik Kim
    • , Sangsu Bae
    • , Jeongbin Park
    •  & Jin-Soo Kim
  3. ToolGen, Inc., Byucksan Kyoungin Digital Valley 2-Cha, Seoul, South Korea.

    • Eunji Kim
    • , Seokjoong Kim
    •  & Hye Ryeong Yu
  4. Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, South Korea.

    • Jinha Hwang
    •  & Jong-Il Kim
  5. Department of Biochemistry, Seoul National University College of Medicine, Seoul, South Korea.

    • Jong-Il Kim
  6. Genomic Medicine Institute (GMI), Medical Research Center, Seoul National University, Seoul, South Korea.

    • Jong-Il Kim

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Contributions

D.K., E.K. and H.R.Y. performed the experiments. D.K., S.B., J.P., J.H. and J.-I.K. performed bioinformatics analyses. J.-S.K. and S.K. supervised the research.

Competing interests

E.K., H.R.Y. and S.K. are employees of ToolGen, Inc. J.-S.K. is a shareholder of ToolGen, Inc.

Corresponding author

Correspondence to Jin-Soo Kim.

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DOI

https://doi.org/10.1038/nmeth.3284