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Nat Methods. 2015 Mar;12(3):237-43, 1 p following 243. doi: 10.1038/nmeth.3284. Epub 2015 Feb 9.

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

Author information

1
1] Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea. [2] Department of Chemistry, Seoul National University, Seoul, South Korea.
2
Department of Chemistry, Seoul National University, Seoul, South Korea.
3
ToolGen, Inc., Byucksan Kyoungin Digital Valley 2-Cha, Seoul, South Korea.
4
Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, South Korea.
5
1] Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, South Korea. [2] Department of Biochemistry, Seoul National University College of Medicine, Seoul, South Korea. [3] Genomic Medicine Institute (GMI), Medical Research Center, Seoul National University, Seoul, South Korea.

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.

PMID:
25664545
DOI:
10.1038/nmeth.3284
[Indexed for MEDLINE]

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