Dual sgRNAs facilitate CRISPR/Cas9-mediated mouse genome targeting
Dual sgRNAs facilitate CRISPR/Cas9-mediated mouse genome targeting
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双 sgRNA 促进 CRISPR/Cas9 介导的小鼠基因组靶向
DOI：
10.1111/febs.12735
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发表时间：
2014-04-01
期刊：
影响因子：
5.4
通讯作者：
Huang, Xingxu
中科院分区：
文献类型：
--
作者：
Zhou, Jiankui;Wang, Jianying;Huang, Xingxu
细菌规律成簇间隔短回文重复序列（CRISPR）/CRISPR相关9（Cas9）系统是一种通用的RNA引导的哺乳动物基因组修饰系统。通过对单细胞期胚胎与Cas9 mRNA和小/单向导（sg）RNA进行共显微注射，实现了小鼠基因组靶向的一步生成。许多研究都集中在提高该系统的效率上。在本研究中，我们报告了同时使用双sgRNA靶向单个基因显著改善了Cas9介导的基因组靶向，双等位基因修饰效率高达78%。我们进一步观察到，靶基因修饰的特征在于有效的种系传递和位点依赖性脱靶效应，并且载脂蛋白E基因敲除介导的血液生化参数缺陷被CRISPR/Cas9介导的遗传基因修饰重演。我们的结果提供了一种双重sgRNA策略，以促进CRISPR/Cas9介导的小鼠基因组靶向。
The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system is a versatile RNA-guided mammalian genome modification system. One-step generation of mouse genome targeting has been achieved by co-microinjection of one-cell stage embryos with Cas9 mRNA and small/single guide (sg)RNA. Many studies have focused on enhancing the efficiency of this system. In the present study, we report that simultaneous use of dual sgRNAs to target an individual gene significantly improved the Cas9-mediated genome targeting with a bi-allelic modification efficiency of up to 78%. We further observed that the target gene modifications were characterized by efficient germline transmission and site-dependent off-target effects, and also that the apolipoprotein E gene knockout-mediated defects in blood biochemical parameters were recapitulated by CRISPR/Cas9-mediated heritable gene modification. Our results provide a dual sgRNAs strategy to facilitate CRISPR/Cas9-mediated mouse genome targeting.