Conditional Knockouts Generated by Engineered CRISPR-Cas9 Endonuclease Reveal the Roles of Coronin in C. elegans Neural Development
Conditional Knockouts Generated by Engineered CRISPR-Cas9 Endonuclease Reveal the Roles of Coronin in C. elegans Neural Development
复制标题
工程化 CRISPR-Cas9 核酸内切酶产生的条件敲除揭示了 Coronin 在秀丽隐杆线虫神经发育中的作用
DOI：
10.1016/j.devcel.2014.07.017
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发表时间：
2014-09-08
影响因子：
11.8
通讯作者：
Ou, Guangshuo
中科院分区：
文献类型：
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作者：
Shen, Zhongfu;Zhang, Xianliang;Ou, Guangshuo
条件基因敲除动物是研究细胞和发育生物学机制的重要工具。我们通过时空操纵RNA引导的DNA内切核酸酶CRISPR-Cas9在秀丽隐杆线虫体细胞谱系中的表达，开发了一种条件性敲除策略。我们表明，这种体细胞CRISPR-Cas9技术提供了一种快速有效的方法，可以在不同发育阶段的各种细胞类型中产生条件性敲除。此外，我们证明了这种方法优于我们最近开发的体细胞TALEN技术，并能够一步产生多个条件性敲除。通过将这些技术与活细胞成像相结合，我们发现一种与人类神经行为功能障碍相关的必需胚胎基因Coronin在C.线虫胚后成神经细胞迁移和轴突发生。我们认为体细胞CRISPR-Cas9平台是唯一适合基于条件基因编辑的生物医学研究的平台。
Conditional gene knockout animals are valuable tools for studying the mechanisms underlying cell and developmental biology. We developed a conditional knockout strategy by spatiotemporally manipulating the expression of an RNA-guided DNA endonuclease, CRISPR-Cas9, in Caenorhabditis elegans somatic cell lineages. We showed that this somatic CRISPR-Cas9 technology provides a quick and efficient approach to generate conditional knockouts in various cell types at different developmental stages. Furthermore, we demonstrated that this method outperforms our recently developed somatic TALEN technique and enables the one-step generation of multiple conditional knockouts. By combining these techniques with live-cell imaging, we showed that an essential embryonic gene, Coronin, which is associated with human neurobehavioral dysfunction, regulates actin organization and cell morphology during C. elegans postembryonic neuroblast migration and neuritogenesis. We propose that the somatic CRISPR-Cas9 platform is uniquely suited for conditional gene editing-based biomedical research.