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

Nature Biotechnology volume 32, pages 670–676 (2014)Cite this article

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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

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Gene Expression Omnibus

References

  1. van der Oost, J., Jore, M.M., Westra, E.R., Lundgren, M. & Brouns, S.J.J. CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem. Sci. 34, 401–407 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Deveau, H., Garneau, J.E. & Moineau, S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu. Rev. Microbiol. 64, 475–493 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Horvath, P. & Barrangou, R. CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167–170 (2010).

    Article  CAS  PubMed  Google Scholar 

  4. Terns, M.P. & Terns, R.M. CRISPR-based adaptive immune systems. Curr. Opin. Microbiol. 14, 321–327 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Marraffini, L.A. & Sontheimer, E.J. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat. Rev. Genet. 11, 181–190 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Mojica, F.J.M., Díez-Villaseñor, C., García-Martínez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Mali, P., Esvelt, K.M. & Church, G.M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gasiunas, G. & Siksnys, V. RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing? Trends Microbiol. 21, 562–567 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Wang, T., Wei, J.J., Sabatini, D.M. & Lander, E.S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014).

    Article  CAS  PubMed  Google Scholar 

  15. Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).

    Article  CAS  PubMed  Google Scholar 

  16. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wu, Y. et al. Correction of a Genetic Disease in Mouse via Use of CRISPR-Cas9. Cell Stem Cell 13, 659–662 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Schwank, G. et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13, 653–658 (2013).

    Article  CAS  PubMed  Google Scholar 

  19. Dickinson, D.J., Ward, J.D., Reiner, D.J. & Goldstein, B. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat. Methods 10, 1028–1034 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Cheng, A.W. et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23, 1163–1171 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Maeder, M.L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Carroll, D. Staying on target with CRISPR-Cas. Nat. Biotechnol. 31, 807–809 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Cradick, T.J., Fine, E.J., Antico, C.J. & Bao, G. CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 41, 9584–9592 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chiu, H., Schwartz, H.T., Antoshechkin, I. & Sternberg, P.W. Transgene-free genome editing in Caenorhabditis elegans using CRISPR-Cas. Genetics 195, 1167–1171 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Cho, S.W. et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24, 132–141 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Sternberg, S.H., Redding, S., Jinek, M., Greene, E.C. & Doudna, J.A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Teytelman, L., Thurtle, D.M., Rine, J. & van Oudenaarden, A. Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins. Proc. Natl. Acad. Sci. USA 110, 18602–18607 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Machanick, P. & Bailey, T.L. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 27, 1696–1697 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Packer, M.J., Dauncey, M.P. & Hunter, C.A. Sequence-dependent DNA structure: tetranucleotide conformational maps. J. Mol. Biol. 295, 85–103 (2000).

    Article  CAS  PubMed  Google Scholar 

  38. Stamatoyannopoulos, J.A. et al. An encyclopedia of mouse DNA elements (Mouse ENCODE). Genome Biol. 13, 418 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Stadler, M.B. et al. DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature 480, 490–495 (2011).

    Article  CAS  PubMed  Google Scholar 

  40. Orioli, A. et al. Widespread occurrence of non-canonical transcription termination by human RNA polymerase III. Nucleic Acids Res. 39, 5499–5512 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Nielsen, S., Yuzenkova, Y. & Zenkin, N. Mechanism of eukaryotic RNA polymerase III transcription termination. Science 340, 1577–1580 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Bartel, D.P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Jinek, M. & Doudna, J.A. A three-dimensional view of the molecular machinery of RNA interference. Nature 457, 405–412 (2009).

    Article  CAS  PubMed  Google Scholar 

  44. Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1247997 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Crooks, G.E., Hon, G., Chandonia, J.-M. & Brenner, S.E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

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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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Author notes

  1. David A Scott and Andrea J Kriz: These authors contributed equally to this work.

Authors and Affiliations

  1. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Xuebing Wu, Anthony C Chiu, Sidi Chen & Phillip A Sharp

  2. Computational and Systems Biology Graduate Program, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Xuebing Wu & Albert W Cheng

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

    David A Scott, Patrick D Hsu, Alexandro E Trevino, Silvana Konermann & Feng Zhang

  4. Department of Brain and Cognitive Sciences, Department of Biological Engineering, McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    David A Scott, Patrick D Hsu, Alexandro E Trevino, Silvana Konermann & Feng Zhang

  5. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Andrea J Kriz, Anthony C Chiu, Daniel B Dadon & Phillip A Sharp

  6. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA

    Patrick D Hsu

  7. Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Daniel B Dadon, Albert W Cheng & Rudolf Jaenisch

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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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