Off-target assessment of CRISPR-Cas9 guiding RNAs in human iPS and mouse ES cells.
The CRISPR-Cas9 system consists of a site-specific, targetable DNA nuclease that holds great potential in gene editing and genome-wide screening applications. To apply the CRISPR-Cas9 system to these assays successfully, the rate at which Cas9 induces DNA breaks at undesired loci must be understood. We characterized the rate of Cas9 off-target activity in typical Cas9 experiments in two human and one mouse cell lines. We analyzed the Cas9 cutting activity of 12 gRNAs in both their targeted sites and ∼90 predicted off-target sites per gRNA. In a Cas9-based knockout experiment, gRNAs induced detectable Cas9 cutting activity in all on-target sites and in only a few off-target sites genome-wide in human 293FT, human-induced pluripotent stem (hiPS) cells, and mouse embryonic stem (ES) cells. Both the cutting rates and DNA repair patterns were highly correlated between the two human cell lines in both on-target and off-target sites. In clonal Cas9 cutting analysis in mouse ES cells, biallelic Cas9 cutting was observed with low off-target activity. Our results show that off-target activity of Cas9 is low and predictable by the degree of sequence identity between the gRNA and a potential off-target site. Off-target Cas9 activity can be minimized by selecting gRNAs with few off-target sites of near complementarity.
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.
E-Pien
EP
Tan
Yilong
Y
Li
Martin Del Castillo
Mdel C
Velasco-Herrera
Kosuke
K
Yusa
Allan
A
Bradley
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D053837:DNA Cleavage; D004260:DNA Repair; D053595:Embryonic Stem Cells / Q000502:physiology*; D004720:Endonucleases / Q000235:genetics; D018390:Gene Targeting; D005818:Genetic Engineering; D057809:HEK293 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology*; D008297:Male; D051379:Mice; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*
D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D000076987:CRISPR-Associated Protein 9; C000600049:Cas9 protein, Francisella novicida; D004720:Endonucleases
genetics; mammal; organism; process