한빛사 논문
Abstract
Daesik Kim1,2,8, Sangsu Bae1,2,7,8, Jeongbin Park2, Eunji Kim3, Seokjoong Kim3, Hye Ryeong Yu3, Jinha Hwang4, Jong-Il Kim4.6 & Jin-Soo Kim1,2
1Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea. 2Department of Chemistry, Seoul National University, Seoul, South Korea. 3ToolGen, Inc., Byucksan Kyoungin Digital Valley 2-Cha, Seoul, South Korea. 4Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, South Korea. 5Department of Biochemistry, Seoul National University College of Medicine, Seoul, South Korea. 6Genomic Medicine Institute (GMI), Medical Research Center, Seoul National University, Seoul, South Korea. 7Present address: Department of Chemistry, Hanyang University, Seoul, South Korea. 8These authors contributed equally to this work.
Correspondence to: Jin-Soo Kim
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.
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