Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

DNA targeting specificity of RNA-guided Cas9 nucleases

This article has been updated

Abstract

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing1,2,3,4,5,6,7,8,9,10. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Optimization of guide RNA architecture for SpCas9-mediated mammalian genome editing.
Figure 2: Single-nucleotide specificity of SpCas9.
Figure 3: Multiple mismatch specificity of SpCas9.
Figure 4: SpCas9-mediated indel frequencies at predicted genomic off-target loci.

Similar content being viewed by others

Accession codes

Primary accessions

BioProject

Change history

  • 28 August 2013

    In the version of this article initially published, funding information was left out of the acknowledgments section. The error has been corrected in the HTML and PDF versions of the article.

References

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 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 

  3. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  6. Cho, S.W., Kim, S., Kim, J.M. & Kim, J.S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).

    CAS  PubMed  Google Scholar 

  7. Chang, N. et al. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res. 23, 465–472 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hwang, W.Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227–229 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Shen, B. et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 23, 720–723 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Gratz, S.J. et al. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics doi:10.1534/genetics.113.152710 (2 July 2013).

  11. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 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 

  13. Guschin, D.Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247–256 (2010).

    Article  CAS  PubMed  Google Scholar 

  14. Bogenhagen, D.F. & Brown, D.D. Nucleotide sequences in Xenopus 5S DNA required for transcription termination. Cell 24, 261–270 (1981).

    Article  CAS  PubMed  Google Scholar 

  15. Bultmann, S. et al. Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers. Nucleic Acids Res. 40, 5368–5377 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Valton, J. et al. Overcoming transcription activator-like effector (TALE) DNA binding domain sensitivity to cytosine methylation. J. Biol. Chem. 287, 38427–38432 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757–761 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Miller, J.C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148 (2011).

    Article  CAS  PubMed  Google Scholar 

  19. Mussolino, C. et al. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res. 39, 9283–9293 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hsu, P.D. & Zhang, F. Dissecting neural function using targeted genome engineering technologies. ACS Chem. Neurosci. 3, 603–610 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Sanjana, N.E. et al. A transcription activator-like effector toolbox for genome engineering. Nat. Protoc. 7, 171–192 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Porteus, M.H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003).

    Article  PubMed  Google Scholar 

  23. Miller, J.C. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25, 778–785 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Sander, J.D. et al. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat. Methods 8, 67–69 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Wood, A.J. et al. Targeted genome editing across species using ZFNs and TALENs. Science 333, 307 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bobis-Wozowicz, S., Osiak, A., Rahman, S.H. & Cathomen, T. Targeted genome editing in pluripotent stem cells using zinc-finger nucleases. Methods 53, 339–346 (2011).

    Article  CAS  PubMed  Google Scholar 

  27. 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 

  28. Michaelis, L.M. Maud “Die kinetik der invertinwirkung.”. Biochemistry Zeitung 49, 333–369 (1913).

    CAS  Google Scholar 

  29. Mahfouz, M.M. et al. De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks. Proc. Natl. Acad. Sci. USA 108, 2623–2628 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank A. Shalek, E. Stamenova and D. Gray for expert help with DNA sequencing, R. Barretto for genome-wide PAM analysis, as well as D. Altshuler, P.A. Sharp, and the entire Zhang Lab for their support and advice. P.D.H. is a James Mills Pierce Fellow. D.A.S. is an National Science Foundation pre-doctoral fellow and J.A.W. is supported by a Life Science Research Foundation Fellowship. X.W. is a Howard Hughes Medical Institute International Student Research Fellow and is supported by National Institutes of Health (NIH) grants R01-GM34277 and R01-CA133404 to P.A. Sharp, X.W.'s thesis advisor. G.B. is supported by an NIH Nanomedicine Development Center Award (PN2EY018244).This work is supported by an NIH Director's Pioneer Award (DP1-MH100706), an NIH Transformative R01 grant (R01-DK097768) to D. Altshuler, the Keck, McKnight, Damon Runyon, Searle Scholars, Klingenstein and Simons Foundations, and Bob Metcalfe and Jane Pauley. The authors wish to dedicate this paper to the memory of Officer Sean Collier, for his caring service to the MIT community and for his sacrifice. Reagents are available to the academic community through Addgene, and associated protocols, support forums and computational tools are available through the Zhang lab website (http://www.genome-engineering.org/).

Author information

Authors and Affiliations

Authors

Contributions

J.A.W. and F.A.R. contributed equally to this work. P.D.H., D.A.S., F.A.R., S.K. and F.Z. designed and performed the experiments. P.D.H., D.A.S., J.A.W., Y.L., S.K., F.A.R. and F.Z. analyzed the data. V.A. and O.S. contributed computational prediction of CRISPR off-target sites and X.W. performed the northern blot analysis. P.D.H., F.A.R., D.A.S. and F.Z. wrote the manuscript with help from all authors.

Corresponding author

Correspondence to Feng Zhang.

Ethics declarations

Competing interests

A patent application has been filed relating to this work, and the authors plan on making the reagents widely available to the academic community through Addgene and to provide software tools via the Zhang lab website (http://www.genome-engineering.org/).

Supplementary information

Supplementary Text and Figures

Supplementary Sequences, Supplementary Figures 1–11 and Supplementary Tables 1–4 (PDF 1235 kb)

Supplementary Table 5

All sequencing data for Figure 2 (XLSX 1740 kb)

Supplementary Table 6

All sequencing data for Figure 3 (XLSX 110 kb)

Supplementary Table 7

All sequencing data for Figure 4 (XLSX 142 kb)

Supplementary Table 8

All sequencing data for expanded set of candidate genomic off-target loci for EMX1 targets 1, 2, 3, and 6 (XLSX 94 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hsu, P., Scott, D., Weinstein, J. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31, 827–832 (2013). https://doi.org/10.1038/nbt.2647

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2647

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing