title: Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence.
abstract: Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a "core" sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease. Our study also found that, single or multiple target mismatches at this region abolished off-target cleavage mediated by active sgRNAs, thus proposing a principle for gene-specific sgRNA design. Characterization of a mismatch sensitive "core" sequence not only enhances our understanding of how this elegant system functions, but also facilitates our efforts to improve targeting specificity of a sgRNA.
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Zheng
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: Yingzi
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Biomics Biotechnologies co. Ltd, Nantong 226016, Jiangsu Province, China.|Small RNA Technology and Application Institute, Nantong University, Nantong 226016, Jiangsu Province, China.
authors.forename: Pingjing
authors.initials: P
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: Zhenxi
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: Letian
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: Leilei
authors.initials: L
authors.lastname: Niu
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: Da
authors.initials: D
authors.lastname: Liang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Yi
authors.affiliation: Biomics Biotechnologies co. Ltd, Nantong 226016, Jiangsu Province, China.|Small RNA Technology and Application Institute, Nantong University, Nantong 226016, Jiangsu Province, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Peng
authors.affiliation: Biomics Biotechnologies co. Ltd, Nantong 226016, Jiangsu Province, China.|Small RNA Technology and Application Institute, Nantong University, Nantong 226016, Jiangsu Province, China.
authors.forename: Wenjian
authors.initials: W
authors.lastname: Feng
authors.affiliation: Department of Stomatology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Biomics Biotechnologies co. Ltd, Nantong 226016, Jiangsu Province, China.|Small RNA Technology and Application Institute, Nantong University, Nantong 226016, Jiangsu Province, China.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Chen
authors.affiliation: Biomics Biotechnologies co. Ltd, Nantong 226016, Jiangsu Province, China.|Small RNA Technology and Application Institute, Nantong University, Nantong 226016, Jiangsu Province, China.
authors.forename: York Yuanyuan
authors.initials: YY
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: Li-He
authors.initials: LH
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
authors.forename: Quan
authors.initials: Q
authors.lastname: Du
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D005818:Genetic Engineering; D016678:Genome; D006801:Humans; D009154:Mutation; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry* / Q000235:genetics*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems