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Generating mutant Aedes aegypti mosquitoes using the CRISPR/Cas9 system.

STAR protocols (2021-04-27)
Hsing-Han Li, Jian-Chiuan Li, Matthew P Su, Kun-Lin Liu, Chun-Hong Chen
ABSTRACT

Implementation of CRISPR/Cas9 methodologies for mosquito gene editing has not yet become widespread. This protocol details the procedure for Aedes aegypti 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).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Phenol:Chloroform:Isoamyl Alcohol 25:24:1, Saturated with 10mM Tris, pH 8.0, 1mM EDTA, for molecular biology
Sigma-Aldrich
Proteinase K from Tritirachium album, lyophilized powder, BioUltra, ≥30 units/mg protein, for molecular biology
Sigma-Aldrich
Ribonuclease A from bovine pancreas, Type I-AS, 50-100 Kunitz units/mg protein