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