High-fidelity endonuclease variant HypaCas9 facilitates accurate allele-specific gene modification in mouse zygotes
High-fidelity endonuclease variant HypaCas9 facilitates accurate allele-specific gene modification in mouse zygotes
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DOI：
10.1038/s42003-019-0627-8
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发表时间：
2019-10-10
影响因子：
5.9
通讯作者：
Naito, Kunihiko
中科院分区：
文献类型：
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作者：
Ikeda, Arisa;Fujii, Wataru;Naito, Kunihiko
CRISPR/Cas9已被广泛用于高效生产转基因动物;然而，该系统可能具有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HypaCas 9在小鼠受精卵中进行精确基因组编辑的有效性。HypaCas 9有效地修饰了靶基因座，同时即使在单核苷酸错配序列中也使脱靶效应最小化。此外，通过将HypaCas 9应用于杂交品系来源的合子中SNP的辨别，我们完成了等位基因特异性基因修饰，并成功地产生了在必需基因中具有单等位基因突变的小鼠。这些结果表明，HypaCas 9的准确性提高有助于产生转基因动物。
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.