A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing.

Autor: Chen P; State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan, China., Zhou J; State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan, China., Wan Y; State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan, China., Liu H; State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan, China., Li Y; State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan, China., Liu Z; State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan, China., Wang H; State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan, China., Lei J; Department of Clinical Oncology, Renmin Hospital of Wuhan University, Wuhan, 430060, China., Zhao K; State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan, China., Zhang Y; Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, 430072, Hubei, People's Republic of China., Wang Y; Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, 430072, Hubei, People's Republic of China., Zhang X; College of Life Sciences, the Institute for Advanced Studies, State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, Wuhan University, Wuhan, 430072, China., Yin L; State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan, China. yinlei@whu.edu.cn.
Jazyk: angličtina
Zdroj: Genome biology [Genome Biol] 2020 Mar 25; Vol. 21 (1), pp. 78. Date of Electronic Publication: 2020 Mar 25.
DOI: 10.1186/s13059-020-01989-2
Abstrakt: Background: AsCas12a and LbCas12a nucleases are reported to be promising tools for genome engineering with protospacer adjacent motif (PAM) TTTV as the optimal. However, the C-containing PAM (CTTV, TCTV, TTCV, etc.) recognition by Cas12a might induce extra off-target edits at these non-canonical PAM sites.
Results: Here, we identify a novel Cas12a nuclease CeCas12a from Coprococcus eutactus, which is a programmable nuclease with genome-editing efficiencies comparable to AsCas12a and LbCas12a in human cells. Moreover, CeCas12a is revealed to be more stringent for PAM recognition in vitro and in vivo followed by very low off-target editing rates in cells. Notably, CeCas12a renders less off-target edits located at C-containing PAM at multiple sites compared to LbCas12a and AsCas12a, as assessed by targeted sequencing methods.
Conclusions: Our study shows that CeCas12a nuclease is active in human cells and the stringency of PAM recognition could be an important factor shaping off-target editing in gene editing. Thus, CeCas12a provides a promising candidate with distinctive characteristics for research and therapeutic applications.
Databáze: MEDLINE
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