Conditional Knockouts Generated by Engineered CRISPR-Cas9 Endonuclease Reveal the Roles of Coronin in C. elegans Neural Development

• Published in: Developmental cell Vol. 30; no. 5; pp. 625 - 636
• Main Authors: Shen, Zhongfu, Zhang, Xianliang, Chai, Yongping, Zhu, Zhiwen, Yi, Peishan, Feng, Guoxin, Li, Wei, Ou, Guangshuo
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.09.2014
• Subjects: Actins - chemistry; Animals; Caenorhabditis elegans - embryology; Cell Lineage; Cell Movement; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; Cytoskeleton - metabolism; Endonucleases - genetics; Gene Expression Regulation, Developmental; Gene Knockout Techniques; Genetic Engineering; Heat-Shock Proteins; Microfilament Proteins - genetics; Mitosis; Mutation; Neurogenesis - physiology; Neurons - physiology
• ISSN: 1534-5807; 1878-1551
• DOI: 10.1016/j.devcel.2014.07.017

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. •Somatic expression of the CRISPR-Cas9 system induces conditional mutations•Rapid one-step generation of conditional mutations of multiple genes•Coronin regulates the actin cytoskeleton and cell morphology in neural development Shen et al. spatiotemporally manipulate CRISPR-Cas9 nuclease expression to create tissue-specific knockouts in C. elegans. The efficiency of nuclease action is sufficient at most gene loci to generate double and triple conditional knockouts. The authors use this method to bypass early lethality and study later neuronal migration events.