Generation of multi-gene knockout rabbits using the Cas9/gRNA system.
Generation of multi-gene knockout rabbits using the Cas9/gRNA system.
复制标题
使用CAS9/GRNA系统生成多基因敲除兔子。
DOI：
10.1186/2045-9769-3-12
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
Lai L
中科院分区：
文献类型：
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作者：
Yan Q;Zhang Q;Yang H;Zou Q;Tang C;Fan N;Lai L
原核生物簇状规则间隔短回文重复序列相关系统(CRISPR)是一种在斑马鱼、小鼠和大鼠等模式生物中进行基因打靶的简单、可靠和有效的技术。在本报告中，我们通过将Cas9 mRNA和引导RNA(GRNA)显微注射到原核期胚胎的细胞质中，将CRISPR技术应用于兔。通过注射1基因(IL2RG)或2基因(IL2RG和RAG1)Cas9mRNA和gRNA获得了双等位基因敲除(KO)兔，效率为100%。我们还对多基因KOS在兔早期胚胎中的效率进行了检测，发现3个基因(IL2RG、RAG1和RAG2)和5个基因(IL2RG、RAG1、RAG2、TIKI1和ALB)在靶点的同时突变效率高达100%。我们的结果表明，Cas9/gRNA系统不仅是一种高效、快速的单基因编辑工具，而且也是一种用于多基因编辑的工具。本文的在线版本(DOI：10.1186/20459769-3-12)包含补充材料，可供授权用户使用。
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. The online version of this article (doi:10.1186/2045-9769-3-12) contains supplementary material, which is available to authorized users.