利用Cas9/gRNA系统生成多基因敲除兔。
Generation of multi-gene knockout rabbits using the Cas9/gRNA system.
作者信息
Yan Quanmei, Zhang Quanjun, Yang Huaqiang, Zou Qingjian, Tang Chengcheng, Fan Nana, Lai Liangxue
机构信息
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.
出版信息
Cell Regen. 2014 Sep 27;3(1):12. doi: 10.1186/2045-9769-3-12. eCollection 2014.
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.
原核生物成簇规律间隔短回文重复序列（CRISPR）相关系统（Cas）是一种用于在斑马鱼、小鼠和大鼠等模式生物中进行基因靶向的简单、强大且高效的技术。在本报告中，我们通过将Cas9信使核糖核酸（mRNA）和引导核糖核酸（gRNA）显微注射到原核期胚胎的细胞质中，将CRISPR技术应用于兔子。通过注射1个基因（IL2rg）或2个基因（IL2rg和RAG1）的Cas9 mRNA和gRNA，我们获得了双等位基因敲除（KO）兔子，效率为100%。我们还测试了早期兔胚胎中多个基因敲除的效率，发现对于3个基因（IL2rg、RAG1和RAG2），靶位点同时基因突变的效率高达100%，对于5个基因（IL2rg、RAG1、RAG2、TIKI1和ALB）为33.3%。我们的结果表明，Cas9/gRNA系统不仅是一种用于兔子单基因编辑的高效快速工具，也是用于多基因编辑的高效快速工具。
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