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

被引：160

作者：

Zhang, Yilan ^{[1
,2
,3
]}

Ge, Xianglian ^{[1
,2
,3
]}

Yang, Fayu ^{[1
,2
,3
]}

Zhang, Liping ^{[1
,2
,3
]}

Zheng, Jiayong ^{[4
]}

Tan, Xuefang ^{[5
]}

Jin, Zi-Bing ^{[1
,2
,3
]}

Qu, Jia ^{[1
,2
,3
]}

Gu, Feng ^{[1
,2
,3
]}

机构：

[1] WenzhouMed Univ, Hosp Eye, Sch Ophthalmol & Optometry, State Key Lab Cultivat Base, Wenzhou 325027, Zhejiang, Peoples R China

[2] Minist Hlth, Key Lab Vision Sci, Wenzhou 325027, Zhejiang, Peoples R China

[3] Zhejiang Prov Key Lab Ophthalmol & Optometry, Wenzhou 325027, Zhejiang, Peoples R China

[4] Peoples Hosp Wenzhou, Dept Gynecol & Obstet, Wenzhou 325000, Zhejiang, Peoples R China

[5] Chinese Acad Sci, Ctr Infect & Immun, GIBH, Guangzhou 510530, Guangdong, Peoples R China

来源：

SCIENTIFIC REPORTS | 2014年 / 4卷

关键词：

CRISPR; CAS9; GENERATION; MOUSE;

D O I：

10.1038/srep05405

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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 NGAPAM 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.

引用

页数：5

共 19 条

[1] Multiplex Genome Engineering Using CRISPR/Cas Systems [J].