Generating Mutant Aedes Aegypti Mosquitoes Using the CRISPR/Cas9 System

Implementation of CRISPR/Cas9 methodologies for mosquito gene editing has not yet become widespread. This protocol details the procedure for Aedes ae-gypti mosquito gene editing using homology-directed repair and fluorescent marker insertion, which facilitates the generation and screening of mutant mos-quito lines for gene function testing. We describe optimized methods for single guide RNA plasmid preparation, homologous recombination donor plasmid con-struction, 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).