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.

Identifying genome-wide off-target sites of CRISPR RNA–guided nucleases and deaminases with Digenome-seq

Abstract

Digested genome sequencing (Digenome-seq) is a highly sensitive, easy-to-carry-out, cell-free method for experimentally identifying genome-wide off-target sites of programmable nucleases and deaminases (also known as base editors). Genomic DNA is digested in vitro using clustered regularly interspaced short palindromic repeats ribonucleoproteins (RNPs; plus DNA-modifying enzymes to cleave both strands of DNA at sites containing deaminated base products, in the case of base editors) and subjected to whole-genome sequencing (WGS) with a typical sequencing depth of 30×. A web-based program is available to map in vitro cleavage sites corresponding to on- and off-target sites. Chromatin DNA, in parallel with histone-free genomic DNA, can also be used to account for the effects of chromatin structure on off-target nuclease activity. Digenome-seq is more sensitive and comprehensive than cell-based methods for identifying off-target sites. Unlike other cell-free methods, Digenome-seq does not involve enrichment of DNA ends through PCR amplification. The entire process other than WGS, which takes ~1–2 weeks, including purification and preparation of RNPs, digestion of genomic DNA and bioinformatic analysis after WGS, takes about several weeks.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Digenome-seq workflow.
Fig. 2: Representative Digenome-seq results.

Data availability

The sample sequencing dataset has been deposited in the European Nucleotide Archive database with accession code PRJEB20021.

Code availability

The source code used to generate Digenome version 2.0 can be accessed at https://github.com/chizksh/digenome-toolkit2 or http://www.rgenome.net/digenome-js.

References

  1. 1.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

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

    PubMed  PubMed Central  Google Scholar 

  3. 3.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    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 

  5. 5.

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

    CAS  PubMed  Google Scholar 

  6. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    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  PubMed  PubMed Central  Google Scholar 

  10. 10.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Morgens, D. W. et al. Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens. Nat. Commun. 8, 15178 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

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

    CAS  PubMed  Google Scholar 

  13. 13.

    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  PubMed  PubMed Central  Google Scholar 

  14. 14.

    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  PubMed  PubMed Central  Google Scholar 

  15. 15.

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

    CAS  PubMed  Google Scholar 

  16. 16.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR–Cas system. Cell 163, 759–771 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).

  20. 20.

    Gehrke, J. M. et al. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977–982 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Wang, X. et al. Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion. Nat. Biotechnol. 36, 946–949 (2018).

    CAS  PubMed  Google Scholar 

  22. 22.

    Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Li, X. et al. Base editing with a Cpf1-cytidine deaminase fusion. Nat. Biotechnol. 36, 324–327 (2018).

    CAS  PubMed  Google Scholar 

  24. 24.

    Kleinstiver, B. P. et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276–282 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Kim, D., Kim, D. E., Lee, G., Cho, S. I. & Kim, J. S. Genome-wide target specificity of CRISPR RNA-guided adenine base editors. Nat. Biotechnol. 37, 430–435 (2019).

    CAS  PubMed  Google Scholar 

  26. 26.

    Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019).

    CAS  PubMed  Google Scholar 

  27. 27.

    Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Kim, D. et al. Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat. Biotechnol. 35, 475–480 (2017).

    CAS  PubMed  Google Scholar 

  29. 29.

    Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR–Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015).

    CAS  PubMed  Google Scholar 

  30. 30.

    Kim, D., Kim, S., Kim, S., Park, J. & Kim, J. S. Genome-wide target specificities of CRISPR–Cas9 nucleases revealed by multiplex Digenome-seq. Genome Res. 26, 406–415 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Park, J. et al. Digenome-seq web tool for profiling CRISPR specificity. Nat. Methods 14, 548–549 (2017).

    CAS  PubMed  Google Scholar 

  32. 32.

    Kim, E. et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat. Commun. 8, 14500 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Kim, D. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat. Biotechnol. 34, 863–868 (2016).

    CAS  PubMed  Google Scholar 

  34. 34.

    Liang, P. et al. Genome-wide profiling of adenine base editor specificity by EndoV-seq. Nat. Commun. 10, 67 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

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

    CAS  PubMed  Google Scholar 

  36. 36.

    Tsai, S. Q. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nat. Methods 14, 607–614 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Cameron, P. et al. Mapping the genomic landscape of CRISPR–Cas9 cleavage. Nat. Methods 14, 600–606 (2017).

    CAS  PubMed  Google Scholar 

  38. 38.

    Kim, D. & Kim, J. DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA. Genome Res. 28, 1894–1900 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Kim, D., Luk, K., Wolfe, S. A. & Kim, J. S. Evaluating and enhancing target specificity of gene-editing nucleases and deaminases. Annu. Rev. Biochem. 88, 191–220 (2019).

    CAS  PubMed  Google Scholar 

  40. 40.

    Tsai, S. Q. & Joung, J. K. Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases. Nat. Rev. Genet. 17, 300–312 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Koo, T., Lee, J. & Kim, J. S. Measuring and reducing off-target activities of programmable nucleases including CRISPR–Cas9. Mol. Cells 38, 475–481 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    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 30, 1473–1475 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Singh, R., Kuscu, C., Quinlan, A., Qi, Y. & Adli, M. Cas9-chromatin binding information enables more accurate CRISPR off-target prediction. Nucleic Acids Res. 43, e118 (2015).

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Xiao, A. et al. CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics 30, 1180–1182 (2014).

    CAS  PubMed  Google Scholar 

  45. 45.

    Montague, T. G., Cruz, J. M., Gagnon, J. A., Church, G. M. & Valen, E. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42, W401–W407 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17, 148 (2016).

    PubMed  PubMed Central  Google Scholar 

  47. 47.

    Wang, X. et al. Unbiased detection of off-target cleavage by CRISPR–Cas9 and TALENs using integrase-defective lentiviral vectors. Nat. Biotechnol. 33, 175–178 (2015).

    CAS  PubMed  Google Scholar 

  48. 48.

    Frock, R. L. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. 33, 179–186 (2015).

    CAS  PubMed  Google Scholar 

  49. 49.

    Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Yan, W. X. et al. BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks. Nat. Commun. 8, 15058 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Wienert, B. et al. Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq. Science 364, 286–289 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Rees, H. A. et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat. Commun. 8, 15790 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

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

    CAS  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. and D.K., the ‘KRIBB Research Initiative Program’ to D.K. and the ‘R&D Convergence Program’ of the National Research Council of Science & Technology (CAP-15-03-KRIBB) to D.K.

Author information

Affiliations

Authors

Contributions

J.-S.K. supervised the research. J.-S.K., D. K. and B.-C.K. wrote the manuscript.

Corresponding author

Correspondence to Jin-Soo Kim.

Ethics declarations

Competing interests

J.-S.K. and D. K. have filed a patent application based on this work. J.-S.K. is a cofounder of, and holds stock in, ToolGen.

Additional information

Peer review information Nature Protocols thanks Vikram Pattanayak and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Kim, D. et al. Nat. Methods 14, 237–243 (2015): https://doi.org/10.1038/nmeth.3284

Kim, D. et al. Nat. Biotechnol. 35, 475–480 (2017): https://doi.org/10.1038/nbt.3852

Kim, D. et al. Nat. Biotechnol. 37, 430–435 (2019): https://doi.org/10.1038/s41587-019-0050-1

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kim, D., Kang, BC. & Kim, JS. Identifying genome-wide off-target sites of CRISPR RNA–guided nucleases and deaminases with Digenome-seq. Nat Protoc 16, 1170–1192 (2021). https://doi.org/10.1038/s41596-020-00453-6

Download citation

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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