Daesik Kim1,2, Sojung Kim1,2, Sunghyun Kim1, Jeongbin Park3, and Jin-Soo Kim1,2,*
1 Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea.
2 Department of Chemistry, Seoul National University, Seoul 151-747, South Korea.
3 Department of Chemistry, Hanyang University, Seoul, South Korea
* To whom correspondence should be addressed. J.-S.K.
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 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.