CRISPR-Cas9-Mediated Genetic Screening in Mice with Haploid Embryonic Stem Cells Carrying a Guide RNA Library
CRISPR-Cas9-Mediated Genetic Screening in Mice with Haploid Embryonic Stem Cells Carrying a Guide RNA Library
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CRISPR-Cas9介导的对带有引导RNA文库的单倍体胚胎干细胞的小鼠进行遗传筛选
DOI：
10.1016/j.stem.2015.06.005
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发表时间：
2015-08-06
期刊：
影响因子：
23.9
通讯作者：
Li, Jinsong
中科院分区：
文献类型：
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作者：
Zhong, Cuiqing;Yin, Qi;Li, Jinsong
小鼠雄激素性单倍体胚胎干细胞（AG-haESCs）经注入MII卵母细胞后可支持半克隆（SC）胚胎的足月发育，因此在遗传修饰方面具有潜在的应用前景。然而，SC幼崽的低出生率限制了这种方法的实际应用。本研究表明，携带DMRs（差异DNA甲基化区）缺失的AG-haESCs可以有效地支持SC幼崽的产生，DMRs（差异DNA甲基化区）控制着两个父本抑制的印迹基因H19和Gtl2。利用CRISPR-Cas9对这些DKO-AG-haESCs进行体外遗传操作，可以高效地产生携带多种修饰的SC小鼠。此外，用组成性表达的sgRNA文库和Cas9转染DKO-AG-haESCs可以进行功能性诱变筛选。因此，DKO-AG-haESCs是在单代小鼠中引入全生物体突变的有效工具。
Mouse androgenetic haploid embryonic stem cells (AG-haESCs) can support full-term development of semi-cloned (SC) embryos upon injection into MII oocytes and thus have potential applications in genetic modifications. However, the very low birth rate of SC pups limits practical use of this approach. Here, we show that AG-haESCs carrying deletions in the DMRs (differentially DNA methylated regions) controlling two paternally repressed imprinted genes, H19 and Gtl2, can efficiently support the generation of SC pups. Genetic manipulation of these DKO-AG-haESCs in vitro using CRISPR-Cas9 can produce SC mice carrying multiple modifications with high efficiency. Moreover, transfection of DKO-AG-haESCs with a constitutively expressed sgRNA library and Cas9 allows functional mutagenic screening. DKO-AG-haESCs are therefore an effective tool for the introduction of organism-wide mutations in mice in a single generation.