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

By Yilan Zhang, Xianglian Ge, Fayu Yang, Liping Zhang, Jiayong Zheng, X. Tan, Zi-Bing Jin, J. Qu, Feng Gu. 2014

203 citations recorded by the source corpus.

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 human EMX 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 of CCMDC with non-canonical PAMs in human cells. The initial results indicated that the effectiveness of NGA PAM for CCMDC is 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.

Subfields: Molecular Biology, Plant Science

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Citations in the corpus

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity (2018, positive): “The four NGA PAM sites averaged 32±2.4% indels for xCas9 and 20±2.7% for SpCas9, a 1.6-fold increase, consistent with previous reports that NGA can serve as a secondary PAM for SpCas9 31 .”
Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions (2017, background): “In comparison, BE3 showed greatly reduced editing for the same non-NGG target sites (0–11% editing), consistent with the known PAM requirement of SpCas9 (Supplementary Fig.”