title: Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq.
abstract: We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on- and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many false-positive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome.
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea; Department of Chemistry, Seoul National University, Seoul 151-747, South Korea;
authors.forename: Daesik
authors.initials: D
authors.lastname: Kim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea; Department of Chemistry, Seoul National University, Seoul 151-747, South Korea;
authors.forename: Sojung
authors.initials: S
authors.lastname: Kim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea;
authors.forename: Sunghyun
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Hanyang University, Seoul 133-791, South Korea.
authors.forename: Jeongbin
authors.initials: J
authors.lastname: Park
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea; Department of Chemistry, Seoul National University, Seoul 151-747, South Korea;
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
mesh_terms: D001665:Binding Sites; D064113:CRISPR-Cas Systems; D018390:Gene Targeting; D015894:Genome, Human; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D054643:INDEL Mutation; D059645:Mutation Rate; D059372:Nucleotide Motifs; D056510:Position-Specific Scoring Matrices; D011485:Protein Binding; D015203:Reproducibility of Results