Wang Lingbo, Li Min-Yin, Qu Chao, Miao Wan-Ying, Yin Qi, Liao Jiaoyang, Cao Hua-Teng, Huang Min, Wang Kai, Zuo Erwei, Peng Guangdun, Zhang Shu-Xin, Chen Guodong, Li Qing, Tang Ke, Yu Qian, Li Zhoujie, Wong Catherine Cl, Xu Guoliang, Jing Naihe, Yu Xiang, Li Jinsong
State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China.
Institute of Neuroscience and State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China.
Cell Res. 2017 Jun;27(6):815-829. doi: 10.1038/cr.2017.58. Epub 2017 Apr 21.
Studying the early function of essential genes is an important and challenging problem in developmental biology. Here, we established a method for rapidly inducing CRISPR-Cas9-mediated mutations in one blastomere of two-cell stage embryos, termed 2-cell embryo-CRISPR-Cas9 injection (2CC), to study the in vivo function of essential (or unknown) genes in founder chimeric mice. By injecting both Cre mRNA and CRISPR-Cas9 targeting the gene of interest into fluorescent reporter mice, the 2CC method can trace both wild-type and mutant cells at different developmental stages, offering internal control for phenotypic analyses of mutant cells. Using this method, we identified novel functions of the essential gene Tet3 in regulating excitatory and inhibitory synaptic transmission in the developing mouse cerebral cortex. By generating chimeric mutant mice, the 2CC method allows for the rapid screening of gene function in multiple tissues and cell types in founder chimeric mice, significantly expanding the current armamentarium of genetic tools.
研究必需基因的早期功能是发育生物学中的一个重要且具有挑战性的问题。在此，我们建立了一种在二细胞期胚胎的一个卵裂球中快速诱导CRISPR-Cas9介导的突变的方法，称为二细胞胚胎-CRISPR-Cas9注射（2CC），以研究必需（或未知）基因在奠基嵌合小鼠中的体内功能。通过将Cre mRNA和靶向感兴趣基因的CRISPR-Cas9注射到荧光报告小鼠中，2CC方法可以追踪不同发育阶段的野生型和突变细胞，为突变细胞的表型分析提供内部对照。使用这种方法，我们确定了必需基因Tet3在调节发育中的小鼠大脑皮质兴奋性和抑制性突触传递中的新功能。通过生成嵌合突变小鼠，2CC方法允许在奠基嵌合小鼠的多种组织和细胞类型中快速筛选基因功能，显著扩展了当前的遗传工具库。
