title: Generation of eGFP and Cre knockin rats by CRISPR/Cas9.
abstract: The type II bacterial CRISPR/Cas [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)] system is a very valuable genome engineering tool, which has been widely used in genome editing of a variety of organisms. Previously, we generated floxed alleles in rats by CRISPR/Cas9. Here, we successfully use a two-cut strategy with one circular vector, which contains the exogenous cDNAs with homology arm regions, in generating knockin rats at the Trdmt1, Nestin and Cck loci. The efficiency of CRISPR/Cas9-mediated knockin was up to 54%. Furthermore, by crossing the Nestin-Cre rat with the Dnmt3b floxed rat and Cck-Cre with the Dnmt1 floxed rat, we detected Cre/loxP-mediated recombination in the F1 generation of rats. We also show that the knockin alleles were germline transmitted. These results provided a simple and flexible engineering strategy for the establishment of knockin rats.
authors.affiliation: Key Laboratory of Human Disease Comparative Medicine, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing, China.
authors.forename: Yuanwu
authors.initials: Y
authors.lastname: Ma
authors.forename: Jing
authors.initials: J
authors.lastname: Ma
authors.forename: Xu
authors.initials: X
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.forename: Lei
authors.initials: L
authors.lastname: Yu
authors.forename: Yingdong
authors.initials: Y
authors.lastname: Lu
authors.forename: Lin
authors.initials: L
authors.lastname: Bai
authors.forename: Bin
authors.initials: B
authors.lastname: Shen
authors.forename: Xingxu
authors.initials: X
authors.lastname: Huang
authors.forename: Lianfeng
authors.initials: L
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D055879:Gene Knock-In Techniques / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D005854:Germ Cells; D049452:Green Fluorescent Proteins / Q000235:genetics*; D019426:Integrases / Q000235:genetics*; D064231:Nestin / Q000235:genetics; D051381:Rats
chemical_list: C577486:Nes protein, rat; D064231:Nestin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases
keywords: CRISPR/Cas9; Cck; knockin; nestin, rat; trdmt1