CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice.
CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice.
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DOI：
10.1038/srep31666
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发表时间：
2016-08-17
影响因子：
4.6
通讯作者：
Ikawa M
中科院分区：
文献类型：
--
作者：
Oji A;Noda T;Fujihara Y;Miyata H;Kim YJ;Muto M;Nozawa K;Matsumura T;Isotani A;Ikawa M
靶向基因破坏的小鼠可以通过在受精卵中表达单向导RNA（sgRNA）/CAS9复合物来有效地产生。然而，复杂的基因组编辑（如大缺失、点突变和敲入）的有限成功仍有待改进。此外，建立者世代的嵌合现象使这些动物的基因型和表型分析复杂化。在这里，我们表明，两个sgRNA的大缺失以及dsDNA介导的点突变在小鼠胚胎干细胞（ESC）中是有效的。dsDNA介导的基因敲入在ESC中也是可行的。最后，我们产生了嵌合小鼠与双等位基因突变胚胎干细胞的致死基因，Dnajb 13，并分析了它们的表型。不仅脑积水的致死表型被抑制，而且我们还发现Dnajb 13是精子纤毛形成所需的。ESC中的双等位基因组编辑和随后的嵌合分析的组合为整个生物体中的快速基因功能分析提供了有用的工具。
Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals. Here we show that large deletions with two sgRNAs as well as dsDNA-mediated point mutations are efficient in mouse embryonic stem cells (ESCs). The dsDNA-mediated gene knockins are also feasible in ESCs. Finally, we generated chimeric mice with biallelic mutant ESCs for a lethal gene, Dnajb13, and analyzed their phenotypes. Not only was the lethal phenotype of hydrocephalus suppressed, but we also found that Dnajb13 is required for sperm cilia formation. The combination of biallelic genome editing in ESCs and subsequent chimeric analysis provides a useful tool for rapid gene function analysis in the whole organism.