Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells.

CRISPR/Cas9 -mediated DNA cleavage (CCMDC) is becoming increasingly used for efficient genome engineering. Proto-spacer adjacent motif (PAM) adjacent to target sequence is one of the key components in the design of CCMDC strategies. It has been reported that NAG sequences are the predominant non-canonical PAM for CCMDC at the humanEMX locus, but it is not clear whether it is universal at other loci. In the present study, we attempted to use a GFP-reporter system to comprehensively and quantitatively test the efficiency ofCCMDCwith non-canonical PAMs in human cells. The initial results indicated that the effectiveness ofNGAPAMforCCMDCis much higher than that of other 14 PAMs including NAG. Then we further designed another three pairs of NGG, NGA and NAG PAMs at different locations in the GFP gene and investigated the corresponding DNA cleavage efficiency. We observed that one group of NGA PAMs have a relatively higher DNA cleavage efficiency, while the other groups have lower efficiency, compared with the corresponding NAG PAMs. Our study clearly demonstrates that NAG may not be the universally predominant non-canonical PAM for CCMDC in human cells. These findings raise more concerns over off-target effects in CRISPR/Cas9-mediated genome engineering. [ABSTRACT FROM AUTHOR]