title: Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing in rats by inhibiting NHEJ and using Cas9 protein.
abstract: Precise modifications such as site mutation, codon replacement, insertion or precise targeted deletion are needed for studies of accurate gene function. The CRISPR/Cas9 system has been proved as a powerful tool to generate gene knockout and knockin animals. But the homologous recombination (HR)-directed precise genetic modification mediated by CRISPR/Cas9 is relatively lower compared with nonhomologous end-joining (NHEJ) pathway and extremely expected to be improved. Here, in this study 2 strategies were used to increase the precise genetic modification in rats. Scr7, a DNA ligase IV inhibitor, first identified as an anti-cancer compound, and considered as a potential NHEJ inhibitor, was used to increase the HR-mediated precise genetic modification. Meanwhile, the Cas9 protein instead of mRNA was used to save the mRNA to protein translation step to improve the precise modification efficiency. The Fabp2 and Dbndd1 loci were selected to knockin Cre and CreER(T2), respectively. Our result showed that both Scr7 and Cas9 protein can increase the precise modification.
authors.affiliation: a Key Laboratory of Human Disease Comparative Medicine, Ministry of Health , Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences , Beijing , China.
authors.forename: Yuanwu
authors.initials: Y
authors.lastname: Ma
authors.affiliation: a Key Laboratory of Human Disease Comparative Medicine, Ministry of Health , Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences , Beijing , China.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: a Key Laboratory of Human Disease Comparative Medicine, Ministry of Health , Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences , Beijing , China.
authors.forename: Xu
authors.initials: X
authors.lastname: Zhang
authors.affiliation: a Key Laboratory of Human Disease Comparative Medicine, Ministry of Health , Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences , Beijing , China.
authors.forename: Lei
authors.initials: L
authors.lastname: Yu
authors.affiliation: a Key Laboratory of Human Disease Comparative Medicine, Ministry of Health , Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences , Beijing , China.
authors.forename: Wei
authors.initials: W
authors.lastname: Dong
authors.affiliation: a Key Laboratory of Human Disease Comparative Medicine, Ministry of Health , Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences , Beijing , China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Pan
authors.affiliation: a Key Laboratory of Human Disease Comparative Medicine, Ministry of Health , Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences , Beijing , China.
authors.forename: Shan
authors.initials: S
authors.lastname: Gao
authors.affiliation: b School of Life Science and Technology , ShanghaiTech University , Shanghai , China.
authors.forename: Xingxu
authors.initials: X
authors.lastname: Huang
authors.affiliation: a Key Laboratory of Human Disease Comparative Medicine, Ministry of Health , Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences , Beijing , China.
authors.forename: Lianfeng
authors.initials: L
authors.lastname: Zhang
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D059766:DNA End-Joining Repair; D000072669:Gene Editing / Q000379:methods*; D056426:Genetic Loci; D051381:Rats
keywords: CRISPR; cas9; cre; dbndd1; fabp2; homologous recombination (HR); rat