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.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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).
Deveau, H., Garneau, J.E. & Moineau, S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu. Rev. Microbiol. 64, 475–493 (2010).
Horvath, P. & Barrangou, R. CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167–170 (2010).
Terns, M.P. & Terns, R.M. CRISPR-based adaptive immune systems. Curr. Opin. Microbiol. 14, 321–327 (2011).
Marraffini, L.A. & Sontheimer, E.J. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat. Rev. Genet. 11, 181–190 (2010).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).
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).
Mali, P., Esvelt, K.M. & Church, G.M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013).
Gasiunas, G. & Siksnys, V. RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing? Trends Microbiol. 21, 562–567 (2013).
Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).
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).
Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).
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).
Wu, Y. et al. Correction of a Genetic Disease in Mouse via Use of CRISPR-Cas9. Cell Stem Cell 13, 659–662 (2013).
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).
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).
Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).
Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).
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).
Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).
Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).
Maeder, M.L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).
Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).
Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013).
Carroll, D. Staying on target with CRISPR-Cas. Nat. Biotechnol. 31, 807–809 (2013).
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).
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).
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).
Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).
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).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
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).
Machanick, P. & Bailey, T.L. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 27, 1696–1697 (2011).
Packer, M.J., Dauncey, M.P. & Hunter, C.A. Sequence-dependent DNA structure: tetranucleotide conformational maps. J. Mol. Biol. 295, 85–103 (2000).
Stamatoyannopoulos, J.A. et al. An encyclopedia of mouse DNA elements (Mouse ENCODE). Genome Biol. 13, 418 (2012).
Stadler, M.B. et al. DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature 480, 490–495 (2011).
Orioli, A. et al. Widespread occurrence of non-canonical transcription termination by human RNA polymerase III. Nucleic Acids Res. 39, 5499–5512 (2011).
Nielsen, S., Yuzenkova, Y. & Zenkin, N. Mechanism of eukaryotic RNA polymerase III transcription termination. Science 340, 1577–1580 (2013).
Bartel, D.P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009).
Jinek, M. & Doudna, J.A. A three-dimensional view of the molecular machinery of RNA interference. Nature 457, 405–412 (2009).
Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1247997 (2014).
Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).
Crooks, G.E., Hon, G., Chandonia, J.-M. & Brenner, S.E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).
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).
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.
The authors declare no competing financial interests.
About this article
Cite this article
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
Nature Protocols (2021)
CRISPR Tools for Physiology and Cell State Changes: Potential of Transcriptional Engineering and Epigenome Editing
Physiological Reviews (2021)
Human Gene Therapy (2021)
Biotechnology Advances (2021)