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

D Kim, S Bae, J Park, E Kim, S Kim, HR Yu, J Hwang… - Nature …, 2015 - nature.com
D Kim, S Bae, J Park, E Kim, S Kim, HR Yu, J Hwang, JI Kim, JS Kim
Nature methods, 2015nature.com
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 …
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.
nature.com