Generation of eGFP and Cre knockin rats by CRISPR/Cas9. (13th August 2014)

• Record Type: Journal Article
• Authors: Ma, Yuanwu; Ma, Jing; Zhang, Xu; Chen, Wei; Yu, Lei; Lu, Yingdong; Bai, Lin; Shen, Bin; Huang, Xingxu; Zhang, Lianfeng
• Is Part Of: FEBS journal. Volume 281:Number 17(2014)
• Page Start: 3779
• Page End: 3790
• Publication Date: 2014-08-13
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.12935
• Languages: English
• ISSNs: 1742-464X

Abstract

<abstract abstract-type="main" id="febs12935-abs-0001"> <title> <x xml:space="preserve">Abstract</x> </title> <p>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 <italic>Trdmt1</italic>, <italic> Nestin</italic> and <italic>Cck</italic> loci. The efficiency of CRISPR/Cas9‐mediated knockin was up to 54%. Furthermore, by crossing the <italic>Nestin‐Cre</italic> rat with the <italic>Dnmt3b</italic> floxed rat and <italic>Cck‐Cre</italic> with the <italic>Dnmt1</italic> floxed rat, we detected Cre/<italic>loxP</italic>‐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.</p> </abstract>