Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes
Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes
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DOI：
10.1016/j.ymeth.2020.04.007
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发表时间：
2021-06-04
期刊：
影响因子：
4.8
通讯作者：
Sugiyama, Fumihiro
中科院分区：
文献类型：
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作者：
Mizuno-Iijima, Saori;Ayabe, Shinya;Sugiyama, Fumihiro
转基因小鼠模型对于体内基因功能研究和人类疾病研究是必不可少的。可以使用CRISPR-Cas等基因组编辑技术将靶向突变引入小鼠胚胎。虽然可以产生小的indel突变的小鼠，但携带大的缺失或基因片段敲入等位基因的小鼠的产生仍然效率低下。我们将CDT1蛋白的核定位特性引入CRISPR-Cas系统，以高效地生产基因工程小鼠。小鼠CDT1连接的Cas9(Cas9-MC)存在于HEK293T细胞和小鼠胚胎的细胞核中。与标准的Cas9相比，Cas9-MC诱导了双等位基因DMD的完全缺失、富含GC的片段敲入和FLOG等位基因敲入，效率较高。这些结果表明，Cas9-MC是制作携带靶向突变的小鼠模型的有用工具。
Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.