Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq Highly Cited Paper

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Title
Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq
Author(s)
Daesik Kim; Sojung Kim; Sunghyun Kim; Jeongbin Park; Jin-Soo Kim
Publication Date
2016-03
Journal
GENOME RESEARCH, v.26, no.3, pp.406 - 415
Publisher
COLD SPRING HARBOR LAB PRESS
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 falsepositive, 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 below 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.
URI
https://pr.ibs.re.kr/handle/8788114/2402
ISSN
1088-9051
Appears in Collections:
Center for Genome Engineering(유전체 교정 연구단) > Journal Papers (저널논문)
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