TY  - JOUR
A1  - Kim, Daesik
A1  - Kim, Sojung
A1  - Kim, Sunghyun
A1  - Park, Jeongbin
A1  - Kim, Jin-Soo
T1  - Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq
Y1  - 2016/03/01 
JF  - Genome Research 
JO  - Genome Research 
SP  - 406 
EP  - 415 
DO  - 10.1101/gr.199588.115 
VL  - 26 
IS  - 3 
UR  - http://genome.cshlp.org/content/26/3/406.abstract 
N2  - 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. 
ER  -