College of Plant Protection, Hainan University, Haikou, Hainan Province, China.
Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests (Hainan University), Ministry of Education, Haikou, Hainan Province, China.
Methods Mol Biol. 2022;2360:347-366. doi: 10.1007/978-1-0716-1633-8_24.
The CRISPR/Cas9 is being developed as an invaluable system that allows rapid and site-specific genome editing in a wide variety of organisms, including diverse insects. It has been successfully used for gene function annotations of RNAi pathway in insect genomics and will facilitate research on RNAi mechanism. Here, we describe a streamlined method to generate and detect somatic and germline knockout mutations of desired target genes in tephritid pests by injecting mRNA encoding the Cas9 endonuclease and in vitro transcribed single guide RNA (sgRNA) into embryos. Target site selection, sgRNA synthesis, Cas9 synthesis, microinjection, and mutation identification are presented in detail.
CRISPR/Cas9 系统正被开发为一种极具价值的系统，可在包括各种昆虫在内的多种生物体中进行快速和特定于位点的基因组编辑。它已成功用于昆虫基因组学中的 RNAi 途径的基因功能注释，并将促进 RNAi 机制的研究。在这里，我们描述了一种简化的方法，通过将编码 Cas9 内切酶的 mRNA 和体外转录的单指导 RNA（sgRNA）注入胚胎，在实蝇害虫中生成和检测所需靶基因的体细胞核和种系敲除突变。详细介绍了靶位点选择、sgRNA 合成、Cas9 合成、显微注射和突变鉴定。
