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

Cited 4 time in Web of Science Cited 4 time in Scopus
Issue Date
2021-02
Citation
Nature Protocols, Vol.16 No.2
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.
ISSN
1754-2189
URI
https://hdl.handle.net/10371/179114
DOI
https://doi.org/10.1038/s41596-020-00453-6
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