Article | Published:

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

Nature Biotechnology volume 33, pages 187197 (2015) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


Primary accessions

Sequence Read Archive


  1. 1.

    & CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

  2. 2.

    , & Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

  3. 3.

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

  4. 4.

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

  5. 5.

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

  6. 6.

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

  7. 7.

    , , & CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 41, 9584–9592 (2013).

  8. 8.

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

  9. 9.

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

  10. 10.

    & Targeted genomic rearrangements using CRISPR/Cas technology. Nat. Commun. 5, 3728 (2014).

  11. 11.

    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).

  12. 12.

    & What's changed with genome editing? Cell Stem Cell 15, 3–4 (2014).

  13. 13.

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

  14. 14.

    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).

  15. 15.

    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).

  16. 16.

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

  17. 17.

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

  18. 18.

    , , , & Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat. Biotechnol. 32, 677–683 (2014).

  19. 19.

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

  20. 20.

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

  21. 21.

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

  22. 22.

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

  23. 23.

    , , , & RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

  24. 24.

    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).

  25. 25.

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

  26. 26.

    , & E-CRISP: fast CRISPR target site identification. Nat. Methods 11, 122–123 (2014).

  27. 27.

    , , , & Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).

  28. 28.

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

  29. 29.

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

  30. 30.

    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).

  31. 31.

    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).

  32. 32.

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

  33. 33.

    , & Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32, 577–582 (2014).

  34. 34.

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

  35. 35.

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

  36. 36.

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

  37. 37.

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

  38. 38.

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

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

Author notes

    • Shengdar Q Tsai
    •  & Zongli Zheng

    These authors contributed equally to this work.


  1. Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Shengdar Q Tsai
    • , Zongli Zheng
    • , Nhu T Nguyen
    • , Matthew Liebers
    • , Ved V Topkar
    • , Vishal Thapar
    • , Nicolas Wyvekens
    • , Cyd Khayter
    • , A John Iafrate
    • , Long P Le
    • , Martin J Aryee
    •  & J Keith Joung
  2. Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Shengdar Q Tsai
    • , Zongli Zheng
    • , Nhu T Nguyen
    • , Matthew Liebers
    • , Ved V Topkar
    • , Vishal Thapar
    • , Nicolas Wyvekens
    • , Cyd Khayter
    • , A John Iafrate
    • , Long P Le
    • , Martin J Aryee
    •  & J Keith Joung
  3. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.

    • Shengdar Q Tsai
    • , Zongli Zheng
    • , A John Iafrate
    • , Long P Le
    • , Martin J Aryee
    •  & J Keith Joung
  4. Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.

    • Zongli Zheng


  1. Search for Shengdar Q Tsai in:

  2. Search for Zongli Zheng in:

  3. Search for Nhu T Nguyen in:

  4. Search for Matthew Liebers in:

  5. Search for Ved V Topkar in:

  6. Search for Vishal Thapar in:

  7. Search for Nicolas Wyvekens in:

  8. Search for Cyd Khayter in:

  9. Search for A John Iafrate in:

  10. Search for Long P Le in:

  11. Search for Martin J Aryee in:

  12. Search for J Keith Joung in:


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.

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.

Corresponding authors

Correspondence to Shengdar Q Tsai or J Keith Joung.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7, Supplementary Tables 1, 3 and 4, Supplementary Results, Supplementary Discussion and Supplementary Protocol

Excel files

  1. 1.

    Supplementary Table 2

    Genomic locations of all GUIDE-Seq detected RGN-induced cleavage sites

About this article

Publication history





Further reading