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Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease

Nature Biotechnology volume 32, pages 677–683 (2014)Cite this article

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Abstract

RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from ∼10 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide.

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Figure 1: Mapping dCas9 binding sites genome-wide.
Figure 2: Sequence determinants of sgRNA:dCas9 binding to targets.
Figure 3: Preservation of base positions in sgRNA guiding sequence and PAM sequence at off-target sites.
Figure 4: RNA-guided Cas9 off-target binding sites are enriched in open chromatin regions.
Figure 5: Targeted deep sequencing to detect indels mediated by WT Cas9 at dCas9 ChIP-seq off-target binding sites.

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Acknowledgements

The research was funded with a departmental startup fund from the University of Virginia and from an American Cancer Society institutional research grant. We would like to thank A. Quinlan, University of Virginia, for technical help with data analysis and D. Burke, University of Virginia, for his critical readings and comments.

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Author notes

  1. Cem Kuscu and Sevki Arslan: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, Virginia, USA

    Cem Kuscu, Sevki Arslan, Ritambhara Singh, Jeremy Thorpe & Mazhar Adli

  2. Department of Biology, Pamukkale University, Denizli, Turkey

    Sevki Arslan

  3. Department of Computer Science, University of Virginia, Charlottesville, Virginia, USA

    Ritambhara Singh

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  1. Cem Kuscu
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Contributions

M.A. designed the study and wrote the manuscript. C.K. and S.A. performed the experiments. J.T. helped with experiments. R.S., C.K. and S.A. analyzed the data.

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Correspondence to Mazhar Adli.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–7 (PDF 5850 kb)

Supplementary Data

dCas9 binding sites mediated by 12 sgRNAs (sgRNA guiding sequence matched bases at off-targets) sorted according to the MACs14 peak score (XLSX 2767 kb)

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Kuscu, C., Arslan, S., Singh, R. et al. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Biotechnol 32, 677–683 (2014). https://doi.org/10.1038/nbt.2916

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