Browse

Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells

Cited 416 time in Web of Science Cited 428 time in Scopus
Authors
Kim, Daesik; Bae, Sangsu; Park, Jeongbin; Kim, Eunji; Kim, Seokjoong; Yu, Hye Ryeong; Hwang, Jinha; Kim, Jong-Il; Kim, Jin-Soo
Issue Date
2015-03
Citation
Nature Methods, Vol.12 No.3, pp.237-243
Abstract
Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.
ISSN
1548-7091
URI
http://hdl.handle.net/10371/165651
DOI
https://doi.org/10.1038/NMETH.3284
Files in This Item:
There are no files associated with this item.
Appears in Collections:
Seoul National University(서울대학교)Featured Researcher's Articles
  • mendeley

Items in S-Space are protected by copyright, with all rights reserved, unless otherwise indicated.

Browse