Skip to main content

Thank you for visiting 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.

GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases

This article has been updated


CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide, off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint 'hotspots'. Finally, GUIDE-seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced, off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases before clinical use.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Design, optimization and application of the GUIDE-seq method.
Figure 2: Sequences of off-target sites identified by GUIDE-seq for ten RGNs.
Figure 3: Analysis of RGN-induced, off-target sequence characteristics.
Figure 4: Comparisons of GUIDE-seq with computational prediction or ChIP-seq methods for identifying RGN off-target sites.
Figure 5: Large-scale structural alterations induced by RGNs.
Figure 6: GUIDE-seq profiles of RGNs directed by tru-gRNAs.

Accession codes

Primary accessions

Sequence Read Archive

Change history

  • 25 June 2015

    In the version of the supplementary file originally posted online, the primer labels 'Nuclease_off_+_GSP1' and 'Nuclease_off_-_GSP1' were switched in Supplementary Table 4, and the discovery thermocycling conditions were missing from the Supplementary Methods. The errors have been corrected in this file as of 25 June 2015.


  1. Sander, J.D. & Joung, J.K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

    Article  CAS  Google Scholar 

  2. Hsu, P.D., Lander, E.S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. 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  Google Scholar 

  9. Ghezraoui, H. et al. Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. Mol. Cell 55, 829–842 (2014).

    Article  CAS  Google Scholar 

  10. Choi, P.S. & Meyerson, M. Targeted genomic rearrangements using CRISPR/Cas technology. Nat. Commun. 5, 3728 (2014).

    Article  CAS  Google Scholar 

  11. Gostissa, M. et al. IgH class switching exploits a general property of two DNA breaks to be joined in cis over long chromosomal distances. Proc. Natl. Acad. Sci. USA 111, 2644–2649 (2014).

    Article  CAS  Google Scholar 

  12. Tsai, S.Q. & Joung, J.K. What's changed with genome editing? Cell Stem Cell 15, 3–4 (2014).

    Article  CAS  Google Scholar 

  13. Marx, V. Gene editing: how to stay on-target with CRISPR. Nat. Methods 11, 1021–1026 (2014).

    Article  CAS  Google Scholar 

  14. Veres, A. et al. Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell 15, 27–30 (2014).

    Article  CAS  Google Scholar 

  15. Smith, C. et al. Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. Cell Stem Cell 15, 12–13 (2014).

    Article  CAS  Google Scholar 

  16. Duan, J. et al. Genome-wide identification of CRISPR/Cas9 off-targets in human genome. Cell Res. 24, 1009–1012 (2014).

    Article  CAS  Google Scholar 

  17. Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676 (2014).

    Article  CAS  Google Scholar 

  18. Kuscu, C., Arslan, S., Singh, R., Thorpe, J. & Adli, M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat. Biotechnol. 32, 677–683 (2014).

    Article  CAS  Google Scholar 

  19. Cencic, R. et al. Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage. PLoS ONE 9, e109213 (2014).

    Article  Google Scholar 

  20. Orlando, S.J. et al. Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology. Nucleic Acids Res. 38, e152 (2010).

    Article  Google Scholar 

  21. Schmidt, M. et al. High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR). Nat. Methods 4, 1051–1057 (2007).

    Article  CAS  Google Scholar 

  22. Gabriel, R. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat. Biotechnol. 29, 816–823 (2011).

    Article  CAS  Google Scholar 

  23. 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 

  24. Lin, Y. et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 42, 7473–7485 (2014).

    Article  CAS  Google Scholar 

  25. Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  Google Scholar 

  26. Heigwer, F., Kerr, G. & Boutros, M. E-CRISP: fast CRISPR target site identification. Nat. Methods 11, 122–123 (2014).

    Article  CAS  Google Scholar 

  27. Fu, Y., Sander, J.D., Reyon, D., Cascio, V.M. & Joung, J.K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).

    Article  CAS  Google Scholar 

  28. Crosetto, N. et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat. Methods 10, 361–365 (2013).

    Article  CAS  Google Scholar 

  29. Osborn, M.J. et al. TALEN-based gene correction for epidermolysis bullosa. Mol. Ther. 21, 1151–1159 (2013).

    Article  CAS  Google Scholar 

  30. Sander, J.D. et al. In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites. Nucleic Acids Res. 41, e181 (2013).

    Article  CAS  Google Scholar 

  31. Fonfara, I. et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res. 42, 2577–2590 (2014).

    Article  CAS  Google Scholar 

  32. Tsai, S.Q. et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32, 569–576 (2014).

    Article  CAS  Google Scholar 

  33. Guilinger, J.P., Thompson, D.B. & Liu, D.R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32, 577–582 (2014).

    Article  CAS  Google Scholar 

  34. 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  Google Scholar 

  35. Ran, F.A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    Article  CAS  Google Scholar 

  36. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).

    Article  Google Scholar 

  37. Zheng, Z. et al. Anchored multiplex PCR for targeted next-generation sequencing. Nat. Med. 20, 1479–1484 (2014).

    Article  CAS  Google Scholar 

  38. Hoffmann, S. et al. A multi-split mapping algorithm for circular RNA, splicing, trans-splicing and fusion detection. Genome Biol. 15, R34 (2014).

    Article  Google Scholar 

Download references


We thank J. Angstman, B. Kleinstiver, Y. Fu, J. Gehrke and R. Cottman for helpful comments on the manuscript and M. Maeder and J. Foden for technical assistance. This work was funded by a National Institutes of Health (NIH) Director's Pioneer Award (DP1 GM105378), NIH R01 GM088040, NIH R01 AR063070, and the Jim and Ann Orr Massachusetts General Hospital (MGH) Research Scholar Award. S.Q.T. was supported by NIH F32 GM105189. This material is based upon work supported by, or in part by, the US Army Research Laboratory and the US Army Research Office under grant number W911NF-11-2-0056. Links to software and resources for analyzing GUIDE-seq data will be made available at:

Author information

Authors and Affiliations



S.Q.T. and J.K.J. conceived of the GUIDE-seq method. S.Q.T., Z.Z., A.J.I., L.P.L. and J.K.J. planned experiments. S.Q.T., Z.Z., N.T.N., M.L., N.W. and C.K. performed experiments. S.Q.T., Z.Z., V.V.T., V.T. and M.J.A. performed bioinformatics and computational analysis of the data. S.Q.T. and J.K.J. wrote the paper.

Corresponding authors

Correspondence to Shengdar Q Tsai or J Keith Joung.

Ethics declarations

Competing interests

J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Editas Medicine and Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1, 3 and 4, Supplementary Results, Supplementary Discussion and Supplementary Protocol (PDF 5037 kb)

Supplementary Table 2

Genomic locations of all GUIDE-Seq detected RGN-induced cleavage sites (XLSX 53 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tsai, S., Zheng, Z., Nguyen, N. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33, 187–197 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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