Yang Guang, Zhu Tianyu, Lu Zongyang, Li Guanglei, Zhang Hao, Feng Songjie, Liu Yajing, Li Jianan, Zhang Yu, Chen Jia, Guo Xuejiang, Huang Xingxu
School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 211166, China.
Sci Bull (Beijing). 2018 Sep 15;63(17):1101-1107. doi: 10.1016/j.scib.2018.07.002. Epub 2018 Jul 5.
Although CRISPR/Cas9 has been widely used to generate knockout mice, two major limitations remain: the founders usually carry a mixture of genotypes, and mosaicism harboring multiple genotypes. Therefore, it takes a long time to get homozygous mutants. Recently developed base editing (BE) system, which introduces C-to-T conversion without double strand DNA cleavage, has been used to introduce artificial stop codons (i-STOP) to prematurely terminate translation, providing a cleaner strategy for genome engineering. Using this strategy, we generated CD160 KO and VISTA/CD160 double KO mice by microinjection of a single sgRNA targeting CD160 and a mixture of sgRNAs targeting VISTA and CD160, respectively. The BE system induced STOP efficiently in mouse embryos and consequently in founder mice without detectable off-target. Most interestingly, the majority of the mutants harbor same genetic modifications, indicating we generated isogenic single and multiplex gene mutant mice by BE-induced STOP. We also obtained homozygous mutant mouse in F1 mice, demonstrating the accelerated strategy in generating animal models.
尽管CRISPR/Cas9已被广泛用于生成基因敲除小鼠，但仍存在两个主要限制：创始小鼠通常携带多种基因型的混合物，以及存在包含多种基因型的嵌合体现象。因此，获得纯合突变体需要很长时间。最近开发的碱基编辑（BE）系统，可在不切割双链DNA的情况下实现C到T的转换，已被用于引入人工终止密码子（i-STOP）以提前终止翻译，为基因组工程提供了一种更简洁的策略。利用这一策略，我们分别通过显微注射靶向CD160的单个sgRNA以及靶向VISTA和CD160的sgRNA混合物，生成了CD160基因敲除小鼠和VISTA/CD160双基因敲除小鼠。BE系统在小鼠胚胎中高效诱导了终止密码子的产生，进而在创始小鼠中也实现了高效诱导，且未检测到脱靶现象。最有趣的是，大多数突变体具有相同的基因修饰，这表明我们通过BE诱导的终止密码子产生了同基因的单基因和多基因突变小鼠。我们还在F1代小鼠中获得了纯合突变小鼠，证明了在生成动物模型方面该加速策略的有效性。
