title: Generation of multi-gene knockout rabbits using the Cas9/gRNA system.
abstract: The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) is a simple, robust and efficient technique for gene targeting in model organisms such as zebrafish, mice and rats. In this report, we applied CRISPR technology to rabbits by microinjection of Cas9 mRNA and guided RNA (gRNA) into the cytoplasm of pronuclear-stage embryos. We achieved biallelic gene knockout (KO) rabbits by injection of 1 gene (IL2rg) or 2 gene (IL2rg and RAG1) Cas9 mRNA and gRNA with an efficiency of 100%. We also tested the efficiency of multiple gene KOs in early rabbit embryos and found that the efficiency of simultaneous gene mutation on target sites is as high as 100% for 3 genes (IL2rg, RAG1 and RAG2) and 33.3% for 5 genes (IL2rg, RAG1, RAG2, TIKI1 and ALB). Our results demonstrate that the Cas9/gRNA system is a highly efficient and fast tool not only for single-gene editing but also for multi-gene editing in rabbits.
authors.affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.
authors.forename: Quanmei
authors.initials: Q
authors.lastname: Yan
authors.affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.
authors.forename: Quanjun
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.
authors.forename: Huaqiang
authors.initials: H
authors.lastname: Yang
authors.affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.
authors.forename: Qingjian
authors.initials: Q
authors.lastname: Zou
authors.affiliation: College of Animal Science, Jilin University, Changchun, 130062 China.
authors.forename: Chengcheng
authors.initials: C
authors.lastname: Tang
authors.affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.
authors.forename: Nana
authors.initials: N
authors.lastname: Fan
authors.affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China ; College of Animal Science, Jilin University, Changchun, 130062 China.
authors.forename: Liangxue
authors.initials: L
authors.lastname: Lai
keywords: Cas9/gRNA system; Multiple-gene knockout; Rabbits