摘要
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 sg RNA targeting CD160 and a mixture of sg RNAs 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.
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 gen-ome 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.
基金
supported by the National Key R&D Program(2016YFC0905901 to X.H.,2016YFA0503300 to X.G.)
the NSFC(81771641 to X.G.)
Fok Ying Tung Education Foundation(161037 to X.G.)
Local Grants(17JC1420103 to X.H.,SKLRM-K201502 to X.G.)
