Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 Japan.
Commun Biol. 2019 Oct 10;2:371. doi: 10.1038/s42003-019-0627-8. eCollection 2019.
CRISPR/Cas9 has been widely used for the efficient generation of genetically modified animals; however, this system could have unexpected off-target effects. In the present study, we confirmed the validity of a high-fidelity Cas9 variant, HypaCas9, for accurate genome editing in mouse zygotes. HypaCas9 efficiently modified the target locus while minimizing off-target effects even in a single-nucleotide mismatched sequence. Furthermore, by applying HypaCas9 to the discrimination of SNP in hybrid strain-derived zygotes, we accomplished allele-specific gene modifications and successfully generated mice with a monoallelic mutation in an essential gene. These results suggest that the improved accuracy of HypaCas9 facilitates the generation of genetically modified animals.
CRISPR/Cas9 已被广泛用于高效产生基因修饰动物；然而，该系统可能会产生意想不到的脱靶效应。在本研究中，我们证实了高保真 Cas9 变体 HypaCas9 在小鼠受精卵中进行精确基因组编辑的有效性。HypaCas9 可有效地修饰靶基因座，同时将脱靶效应降至最低，即使在单核苷酸错配序列中也是如此。此外，通过将 HypaCas9 应用于杂种来源的受精卵中 SNP 的鉴别，我们实现了等位基因特异性基因修饰，并成功地在一个必需基因中产生了单等位基因突变的小鼠。这些结果表明 HypaCas9 的改进准确性有助于基因修饰动物的产生。
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