title: Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells.
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.
authors: 1] Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea. [2] Department of Chemistry, Seoul National University, Seoul, South Korea.
authors: Daesik
authors: D
authors: Kim
authors: 1] Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea. [2] Department of Chemistry, Seoul National University, Seoul, South Korea.
authors: Sangsu
authors: S
authors: Bae
authors: Department of Chemistry, Seoul National University, Seoul, South Korea.
authors: Jeongbin
authors: 0000000290644912
authors: J
authors: Park
authors: ToolGen, Inc., Byucksan Kyoungin Digital Valley 2-Cha, Seoul, South Korea.
authors: Eunji
authors: E
authors: Kim
authors: ToolGen, Inc., Byucksan Kyoungin Digital Valley 2-Cha, Seoul, South Korea.
authors: Seokjoong
authors: S
authors: Kim
authors: ToolGen, Inc., Byucksan Kyoungin Digital Valley 2-Cha, Seoul, South Korea.
authors: Hye Ryeong
authors: HR
authors: Yu
authors: Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, South Korea.
authors: Jinha
authors: J
authors: Hwang
authors: 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.
authors: Jong-Il
authors: JI
authors: Kim
authors: 1] Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea. [2] Department of Chemistry, Seoul National University, Seoul, South Korea.
authors: Jin-Soo
authors: JS
authors: Kim
mesh_terms: D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D015894:Genome, Human; D006238:Haploidy; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D009154:Mutation; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D004720:Endonucleases