Generating mutant Aedes aegypti mosquitoes using the CRISPR/Cas9 system

Hsing-Han Li; Jian-Chiuan Li; Matthew P Su; Kun-Lin Liu; Chun-Hong Chen

doi:10.1016/j.xpro.2021.100432

Generating mutant Aedes aegypti mosquitoes using the CRISPR/Cas9 system

STAR Protoc. 2021 Apr 8;2(2):100432. doi: 10.1016/j.xpro.2021.100432. eCollection 2021 Jun 18.

Authors

Hsing-Han Li¹, Jian-Chiuan Li¹, Matthew P Su², Kun-Lin Liu³, Chun-Hong Chen^{1

3}

Affiliations

¹ National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli 350401, Taiwan.
² Department of Biological Science, Nagoya University, Nagoya 464-8602, Japan.
³ National Mosquito-Borne Diseases Control Research Center, National Health Research Institutes, Miaoli 350401, Taiwan.

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).

Keywords: CRISPR; Genetics.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Aedes / genetics*
Animals
CRISPR-Cas Systems / genetics*
Female
Gene Editing / methods*
Larva / genetics
Male
Polymerase Chain Reaction
RNA, Guide, CRISPR-Cas Systems / genetics
Recombinational DNA Repair / genetics

Substances

RNA, Guide, CRISPR-Cas Systems