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.

High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity

Nature Biotechnology volume 31, pages 839–843 (2013)Cite this article

Subjects

Abstract

The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 1012 potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: In vitro selection overview.
Figure 2: In vitro selection results for Cas9:CLTA1 sgRNA.

Accession codes

Primary accessions

Sequence Read Archive

References

  1. Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731–734 (2011).

    Article  CAS  Google Scholar 

  2. Zou, J. et al. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell 5, 97–110 (2009).

    Article  CAS  Google Scholar 

  3. Hockemeyer, D. et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat. Biotechnol. 27, 851–857 (2009).

    Article  CAS  Google Scholar 

  4. Doyon, Y. et al. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat. Biotechnol. 26, 702–708 (2008).

    Article  CAS  Google Scholar 

  5. Meng, X., Noyes, M.B., Zhu, L.J., Lawson, N.D. & Wolfe, S.A. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat. Biotechnol. 26, 695–701 (2008).

    Article  CAS  Google Scholar 

  6. Sander, J.D. et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat. Biotechnol. 29, 697–698 (2011).

    Article  CAS  Google Scholar 

  7. Tesson, L. et al. Knockout rats generated by embryo microinjection of TALENs. Nat. Biotechnol. 29, 695–696 (2011).

    Article  CAS  Google Scholar 

  8. Cui, X. et al. Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat. Biotechnol. 29, 64–67 (2011).

    Article  CAS  Google Scholar 

  9. Perez, E.E. et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808–816 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  15. DiCarlo, J.E. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336–4343 (2013).

    Article  CAS  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  Google Scholar 

  17. Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39, 9275–9282 (2011).

    Article  CAS  Google Scholar 

  18. Semenova, E. et al. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl. Acad. Sci. USA 108, 10098–10103 (2011).

    Article  CAS  Google Scholar 

  19. 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  Google Scholar 

  20. Pattanayak, V., Ramirez, C.L., Joung, J.K. & Liu, D.R. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat. Methods 8, 765–770 (2011).

    Article  CAS  Google Scholar 

  21. Doyon, J.B., Pattanayak, V., Meyer, C.B. & Liu, D.R. Directed evolution and substrate specificity profile of homing endonuclease I-SceI. J. Am. Chem. Soc. 128, 2477–2484 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

V.P., J.P.G. and D.R.L. were supported by DARPA HR0011-11-2-0003, DARPA N66001-12-C-4207, and the Howard Hughes Medical Institute. V.P. was supported by award no. T32GM007753 from the National Institute of General Medical Sciences. S.L. and J.A.D. were supported by the Howard Hughes Medical Institute (HHMI); E.M. was supported by US National Institutes of Health grant R01GM073794-05 to J.A.D.; J.A.D. and D.R.L. are HHMI Investigators.

Author information

Authors and Affiliations

  1. Department of Chemistry & Chemical Biology and Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    Vikram Pattanayak, John P Guilinger & David R Liu

  2. Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, California, USA

    Steven Lin, Enbo Ma & Jennifer A Doudna

  3. Department of Chemistry, University of California, Berkeley, California, USA

    Jennifer A Doudna

  4. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Jennifer A Doudna

Authors
  1. Vikram Pattanayak
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Steven Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John P Guilinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Enbo Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jennifer A Doudna
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. David R Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

V.P., S.L., J.P.G. and E.M. performed the experiments, designed the research, analyzed the data and wrote the manuscript. J.A.D. and D.R.L. designed the research, analyzed the data and wrote the manuscript.

Corresponding authors

Correspondence to Jennifer A Doudna or David R Liu.

Ethics declarations

Competing interests

The authors have filed provisional patents related to this work.

Supplementary information

Supplementary Text and Figures

Supplementary Notes, Supplementary Figures 1–26, Supplementary Tables 2–8 and Supplementary Algorithms (PDF 16893 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Pattanayak, V., Lin, S., Guilinger, J. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31, 839–843 (2013). https://doi.org/10.1038/nbt.2673

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2673

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research