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Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells

Abstract

Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.

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Figure 1: Genome-wide in vivo binding of dCas9-sgRNA.
Figure 2: A 5-nucleotide seed for dCas9 binding.
Figure 3: Chromatin accessibility is a major determinant of binding in vivo.
Figure 4: Seed sequences influence sgRNA abundance and specificity.
Figure 5: Indel frequencies at on-target sites and 295 off-target sites.
Figure 6: A model for Cas9 target binding and cleavage.

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Gene Expression Omnibus

Sequence Read Archive

Referenced accessions

Gene Expression Omnibus

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Acknowledgements

We would like to thank J. Zamudio and T. Kelly for optimizing the ChIP protocol, and the entire Sharp lab for support and discussion. We also thank the Core Facility in the Swanson Biotechnology Center at the David H. Koch Institute for Integrative Cancer Research at MIT for their assistance with high-throughput sequencing. This work was supported by United States Public Health Service grants RO1-GM34277, R01-CA133404 from the National Institutes of Health, and PO1-CA42063 from the National Cancer Institute to P.A.S., and partially by Cancer Center Support (core) grant P30-CA14051 from the National Cancer Institute. F.Z. is supported by an US National Institutes of Health Director's Pioneer Award (1DP1-MH100706), the Keck, McKnight, Poitras, Merkin, Vallee, Damon Runyon, Searle Scholars, Klingenstein, and Simons Foundations, Bob Metcalfe, and Jane Pauley. X.W. is a Howard Hughes Medical Institute International Student Research Fellow. S.C. is a Damon Runyon Fellow (DRG-2117-12). P.D.H. is a James Mills Pierce Fellow. D.A.S. is an US National Science Foundation pre-doctoral fellow.

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Contributions

X.W., F.Z. and P.A.S. designed experiments; X.W. and A.J.K. performed most experiments; D.A.S. performed targeted indel sequencing; A.W.C. and D.B.D. cloned the piggyBac dCas9 and sgRNA expressing vectors; A.C.C. generated the dCas9 stable cell line; P.D.H., A.E.T. and S.K. purified Cas9; P.D.H. contributed to in vitro binding assay; S.C. contributed to ChIP experiments with transient transfection. X.W., F.Z. and P.A.S. wrote the manuscript with help from all other authors. R.J., F.Z. and P.A.S. supervised the research.

Corresponding authors

Correspondence to Feng Zhang or Phillip A Sharp.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 (PDF 1054 kb)

Supplementary Table 1

ChIP peaks identified for four sgRNAs. (XLSX 776 kb)

Supplementary Table 2 (XLSX 30 kb)

Supplementary Table 3 (XLSX 110 kb)

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Wu, X., Scott, D., Kriz, A. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat Biotechnol 32, 670–676 (2014). https://doi.org/10.1038/nbt.2889

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