National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli 350401, Taiwan.
Department of Biological Science, Nagoya University, Nagoya 464-8602, Japan.
STAR Protoc. 2021 Apr 8;2(2):100432. doi: 10.1016/j.xpro.2021.100432. eCollection 2021 Jun 18.
Implementation of CRISPR/Cas9 methodologies for mosquito gene editing has not yet become widespread. This protocol details the procedure for mosquito gene editing using homology-directed repair and fluorescent marker insertion, which facilitates the generation and screening of mutant mosquito lines for gene function testing. We describe optimized methods for single guide RNA plasmid preparation, homologous recombination donor plasmid construction, embryo microinjection, and precise gene knock-in confirmation. We also provide general guidance for establishing mutant mosquito lines. For details on the practical use and execution of this protocol, please refer to Li et al. (2020).
CRISPR/Cas9 方法在蚊子基因编辑中的应用尚未广泛普及。本方案详细介绍了利用同源重组修复和荧光标记插入进行蚊子基因编辑的步骤，这有助于生成和筛选具有突变体的蚊子品系，以进行基因功能测试。我们描述了优化的单向导 RNA 质粒制备、同源重组供体质粒构建、胚胎显微注射和精确基因敲入确认方法。我们还为建立突变体蚊子品系提供了一般性指导。有关本方案实际使用和执行的详细信息，请参考 Li 等人（2020 年）的文献。
