Optimized CRISPR/Cas9-mediated in vivo genome engineering applicable to monitoring dynamics of endogenous proteins in the mouse neural tissues
Optimized CRISPR/Cas9-mediated in vivo genome engineering applicable to monitoring dynamics of endogenous proteins in the mouse neural tissues
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DOI：
10.1038/s41598-019-47721-4
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发表时间：
2019-08-05
影响因子：
4.6
通讯作者：
Oinuma, Izumi
中科院分区：
文献类型：
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作者：
Matsuda, Takahiko;Oinuma, Izumi
为了分析靶蛋白的原位表达、定位和功能动力学，特别是在活细胞中，开发一种方便、通用、高效的方法来精确地将外源基因引入基因组，这将适用于内源蛋白的标记和工程。通过将CRISPR/Cas9基因组编辑技术与电穿孔技术相结合，我们成功地在发育中的小鼠视网膜和大脑的神经元和神经胶质细胞中创建了敲入等位基因，由此产生了GFP （RFP）标记的内源性蛋白。通过单细胞基因分型和Western blot分析证实基因靶向正确。成功地高效靶向了多个基因位点。此外，我们成功地利用一种自切2A肽，从内源基因位点改造小鼠基因组，表达外源基因。我们的方法可以在单细胞分辨率下监测内源蛋白定位和mRNA和蛋白表达水平的生理变化。这项工作揭示了一种强大而广泛适用的方法，用于可视化和操纵神经组织中的内源性蛋白质。
To analyze the expression, localization, and functional dynamics of target proteins in situ, especially in living cells, it is important to develop a convenient, versatile, and efficient method to precisely introduce exogenous genes into the genome, which is applicable for labeling and engineering of the endogenous proteins of interest. By combining the CRISPR/Cas9 genome editing technology with an electroporation technique, we succeeded in creating knock-in alleles, from which GFP (RFP)-tagged endogenous proteins are produced, in neurons and glial cells in vivo in the developing mouse retina and brain. Correct gene targeting was confirmed by single-cell genotyping and Western blot analysis. Several gene loci were successfully targeted with high efficiency. Moreover, we succeeded in engineering the mouse genome to express foreign genes from the endogenous gene loci using a self-cleaving 2A peptide. Our method could be used to monitor the physiological changes in localization of endogenous proteins and expression levels of both mRNA and protein at a single cell resolution. This work discloses a powerful and widely applicable approach for visualization and manipulation of endogenous proteins in neural tissues.