Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100059, China.
Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
STAR Protoc. 2023 Dec 15;4(4):102720. doi: 10.1016/j.xpro.2023.102720. Epub 2023 Nov 17.
Gene silencing by P-element-induced wimpy testis-interacting RNAs is a mechanism to maintain genome integrity in germ cells. Here, we present a protocol for knockin or knockout editing of male germline genome mediated by CRISPR-Cas9 technology in Caenorhabditis elegans. We describe steps for constructing edited plasmids, microinjecting worms with these plasmids, and screening edited worms. We then detail procedures for dissecting released sperm and their observation with fluorescence microscopy. Engineered worms provide a model for studying hermaphrodite/male fertility or protein localization in vivo. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021)..
P 元件诱导的软弱睪丸相互作用 RNA 基因沉默是维持生殖细胞基因组完整性的一种机制。在这里，我们提供了一种使用 CRISPR-Cas9 技术在秀丽隐杆线虫中对雄性生殖系基因组进行敲入或敲除编辑的方案。我们描述了构建编辑质粒、用这些质粒微注射线虫以及筛选编辑线虫的步骤。然后，我们详细介绍了分离释放精子的程序，并通过荧光显微镜观察它们。工程化的线虫为研究雌雄同体/雄性育性或蛋白质在体内的定位提供了模型。有关该方案使用和执行的完整详细信息，请参阅 Wang 等人（2021 年）。
