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Kim, Dae Sik
유전체 교정 연구단
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Identifying genome-wide off-target sites of CRISPR RNA-guided nucleases and deaminases with Digenome-seq

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Title
Identifying genome-wide off-target sites of CRISPR RNA-guided nucleases and deaminases with Digenome-seq
Author(s)
Daesik Kim; Beum-Chang Kang; Jin-Soo Kim
Publication Date
2021-02
Journal
NATURE PROTOCOLS, v.16, no.2, pp.1170 - 1192
Publisher
NATURE RESEARCH
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 30x. 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 similar to 1-2 weeks, including purification and preparation of RNPs, digestion of genomic DNA and bioinformatic analysis after WGS, takes about several weeks.
URI
https://pr.ibs.re.kr/handle/8788114/9502
DOI
10.1038/s41596-020-00453-6
ISSN
1754-2189
Appears in Collections:
Center for Genome Engineering(유전체 교정 연구단) > 1. Journal Papers (저널논문)
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