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，但仍然存在两个主要限制：创始人通常携带混合基因型，以及包含多种基因型的嵌合体。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) 系统引入了无需双链 DNA 切割的 C-to-T 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更清洁的策略。使用这种策略，我们通过显微注射靶向CD160的单个 sgRNA和靶向VISTA和CD160的 sgRNA 混合物来生成CD160 KO 和VISTA / CD160双 KO 小鼠， 分别。BE 系统在小鼠胚胎中有效地诱导了 STOP，因此在没有检测到脱靶的情况下在创始人小鼠中诱导了 STOP。最有趣的是，大多数突变体具有相同的基因修饰，表明我们通过 BE 诱导的 STOP 生成了同基因单基因和多重基因突变小鼠。我们还在 F1 小鼠中获得了纯合突变小鼠，证明了生成动物模型的加速策略。