title: Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells.
abstract: 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.
authors: 1] School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang 325027 China [2].
authors: Yilan
authors: Y
authors: Zhang
authors: 1] School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang 325027 China [2].
authors: Xianglian
authors: X
authors: Ge
authors: School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang 325027 China.
authors: Fayu
authors: F
authors: Yang
authors: School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang 325027 China.
authors: Liping
authors: L
authors: Zhang
authors: Department of Gynecology and Obstetrics, People's Hospital of Wenzhou, Wenzhou, Zhejiang 325000 China.
authors: Jiayong
authors: J
authors: Zheng
authors: Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences, Guangzhou, Guangdong 510530 China.
authors: Xuefang
authors: X
authors: Tan
authors: School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang 325027 China.
authors: Zi-Bing
authors: ZB
authors: Jin
authors: School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang 325027 China.
authors: Jia
authors: J
authors: Qu
authors: School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang 325027 China.
authors: Feng
authors: F
authors: Gu
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D053837:DNA Cleavage; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D059372:Nucleotide Motifs / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
chemical_list: D064130:CRISPR-Associated Proteins; D049452:Green Fluorescent Proteins