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

Affiliation

Kim D(#)(1)(2), Kang BC(#)(1), Kim JS(3)(4).
Author information:
(1)Center for Genome Engineering, Institute for Basic Science
(IBS), Seoul, Republic of Korea.
(2)Genome Editing Research Center, KRIBB, Daejeon, Republic of Korea.
(3)Center for Genome Engineering, Institute for Basic Science
(IBS), Seoul, Republic of Korea. [Email]
(4)Department of Chemistry, Seoul National University, Seoul, Republic of Korea. [Email]
(#)Contributed equally

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.