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.

Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells

Abstract

Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5′ ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Workflow of off-target analysis of gene KO clones via WGS.
Figure 2: RGEN-mediated genomic DNA digestion in vitro.
Figure 3: RGEN-induced 'digenome' sequencing to capture off-target sites.
Figure 4: Off-target sites of the HBB RGEN captured by Digenome-seq and validated by targeted deep sequencing.
Figure 5: Comparison of conventional sgRNAs with modified sgRNAs that include two extra guanine nucleotides.

Accession codes

Primary accessions

Sequence Read Archive

References

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

    Article  PubMed  Google Scholar 

  2. Bibikova, M., Beumer, K., Trautman, J.K. & Carroll, D. Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003).

    CAS  Article  PubMed  Google Scholar 

  3. Urnov, F.D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).

    CAS  Article  PubMed  Google Scholar 

  4. Kim, H.J., Lee, H.J., Kim, H., Cho, S.W. & Kim, J.S. Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Res. 19, 1279–1288 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Kim, H., Um, E., Cho, S.R., Jung, C. & Kim, J.S. Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nat. Methods 8, 941–943 (2011).

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  7. Kim, Y. et al. A library of TAL effector nucleases spanning the human genome. Nat. Biotechnol. 31, 251–258 (2013).

    CAS  Article  PubMed  Google Scholar 

  8. Kim, Y.K. et al. TALEN-based knockout library for human microRNAs. Nat. Struct. Mol. Biol. 20, 1458–1464 (2013).

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  11. 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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Cho, S.W., Lee, J., Carroll, D., Kim, J.S. & Lee, J. Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195, 1177–1180 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 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  Article  PubMed  Google Scholar 

  16. Kim, H. & Kim, J.S. A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15, 321–334 (2014).

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Brunet, E. et al. Chromosomal translocations induced at specified loci in human stem cells. Proc. Natl. Acad. Sci. USA 106, 10620–10625 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Lee, H.J., Kim, E. & Kim, J.S. Targeted chromosomal deletions in human cells using zinc finger nucleases. Genome Res. 20, 81–89 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Lee, H.J., Kweon, J., Kim, E., Kim, S. & Kim, J.S. Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases. Genome Res. 22, 539–548 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Kim, S., Kim, D., Cho, S.W., Kim, J. & Kim, J.S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Ramakrishna, S. et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 24, 1020–1027 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Carette, J.E. et al. Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature 477, 340–343 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. Kim, Y., Kweon, J. & Kim, J.S. TALENs and ZFNs are associated with different mutation signatures. Nat. Methods 10, 185 (2013).

    Article  PubMed  Google Scholar 

  35. Robinson, J.T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. Bae, S., Park, J. & Kim, J.S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 35, 1473–1475 (2014).

    Article  Google Scholar 

  37. Kim, J.M., Kim, D., Kim, S. & Kim, J.S. Genotyping with CRISPR-Cas-derived RNA-guided endonucleases. Nat. Commun. 5, 3157 (2014).

    Article  PubMed  Google Scholar 

  38. 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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  41. 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).

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  44. Frock, R.L. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. doi:10.1038/nbt.3101 (15 December 2014).

  45. Tsai, S.Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. doi:10.1038/nbt.3117 (16 December 2014).

  46. Schmittgen, T.D. & Livak, K.J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108 (2008).

    CAS  Article  PubMed  Google Scholar 

  47. Raczy, C. et al. Isaac: ultra-fast whole-genome secondary analysis on Illumina sequencing platforms. Bioinformatics 29, 2041–2043 (2013).

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

This research was supported by grants from the Institute for Basic Science (IBS-R021-D1) to J.-S.K., Korea Health Industry Development Institute (HI14C1277) to J.-I.K., and Korea Institute of Planning and Evaluation for Technology of Food, Agriculture, Forestry and Fisheries (311011-05-3-SB010) to S.K.

Author information

Authors and Affiliations

Authors

Contributions

D.K., E.K. and H.R.Y. performed the experiments. D.K., S.B., J.P., J.H. and J.-I.K. performed bioinformatics analyses. J.-S.K. and S.K. supervised the research.

Corresponding author

Correspondence to Jin-Soo Kim.

Ethics declarations

Competing interests

E.K., H.R.Y. and S.K. are employees of ToolGen, Inc. J.-S.K. is a shareholder of ToolGen, Inc.

Integrated supplementary information

Supplementary Figure 1 Analysis of gene KO clones via T7E1 assay and DNA sequencing.

(a) T7E1 assay confirming gene knockout in clonal populations of HAP1 haploid cells. WT, wild-type; MT, mutant; WT+MT, a 1:1 mixture of WT and MT PCR amplicons. (b) DNA sequences of wild-type and mutant clones. The PAM is shown in blue. Inserted bases are shown in red.

Supplementary Figure 2 Analysis of off-target effects in gene KO clones via WGS.

(a) Small deletion in the ERBB3 KO clone confirmed by Sanger sequencing. (b) RGEN-mediated mutagenesis at the on-target and potential off-target sites. Mutation frequencies (%) were measured using T7E1 and targeted deep sequencing. (c) The on-target mutant sequence in the FGFR4 KO clone. The PAM sequence is shown in blue and inserted bases are shown in red. (d) Integrative Genomics Viewer (IGV) image at the FGFR4 on-target site.

Supplementary Figure 3 Examination of potential off-target sites.

(a) The number of potential off-target sites that differ from on-target sites by up to 8 nucleotides or by 2 nucleotides with a DNA or RNA bulge of up to 5 nucleotides in length. (b) Schematic of consensus sequence generation. (c) On-target mutations in five KO clones identified by consensus sequence comparison.

Supplementary Figure 4 RGEN-induced digenome sequencing to capture off-target sites.

(a-d) Representative IGV images obtained using the HBB-specific RGEN at the potential off-target sites OT1 (a), OT3 (b), OT7 (c), and OT12 (d). An indel is indicated by an arrow (a) or shown in a box (b).

Supplementary Figure 5 5′-end plot.

(a) An IGV image at a nuclease cleavage site. (b, c) 5’ End plots showing the absolute and relative number of sequence reads with the same 5’ end across nucleotide positions at the OT1 (b) and OT3 (c) sites.

Supplementary Figure 6 False positive positions captured in the intact genome sequences.

(a-c) Representative IGV images around false-positive sites that resulted from naturally-occurring indels in HAP1 cells.

Supplementary Figure 7 Indel sequences induced by the HBB RGEN at newly validated off-target sites.

(a, b) Off-target indels were detected by targeted deep sequencing. Inserted nucleotides are shown in red and the PAM sequence is shown in blue.

Supplementary Figure 8 Off-target sites of the VEGFA RGEN captured by Digenome-seq.

(a) 5’ End plots at the one of VEGF-A off-target site. (b) Heatmap comparing digenome-captured sites with the on-target site. Dark red and dark blue correspond to 100% and 0% matches, respectively, at a given position. (c) Sequence logo obtained via WebLogo using DNA sequences at digenome-captured sites. (d) Summary of Digenome-seq and targeted deep sequencing. N.D., not determined. (e) Off-target sites validated by targeted deep sequencing. Blue and red bars represent indel frequencies obtained using mock-transfected HAP1 cells and the VEGF-A RGEN-transfected HAP1 cells, respectively. (Left) DNA sequences of on-target and off-target sites. Mismatched bases are shown in red. The PAM is shown in blue. (Right) P value was calculated by the Fisher exact test. Additional deep sequencing results can be found in Supplementary Table 3.

Supplementary Figure 9 RGEN-induced digenome sequencing to capture off-target sites of the VEGFA-targeting RGEN.

(a-d) 5’ End plots showing the absolute and relative number of sequence reads with the same 5’ end across nucleotide positions in on-target (a) and off-target region (b-d).

Supplementary Figure 10 Indel sequences induced by the VEGFA RGEN at newly validated off-target sites.

(a-d) Off-target indels were detected by targeted deep sequencing. Inserted nucleotides are shown in red and the PAM sequence is shown in blue.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Tables 1–6 and Supplementary Notes 1 and 2 (PDF 4070 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kim, D., Bae, S., Park, J. et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods 12, 237–243 (2015). https://doi.org/10.1038/nmeth.3284

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.3284

Further reading

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