Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus

Abstract

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR‐associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre‐blastoderm embryos, 30%–50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in G1 pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9‐mediated gene editing system can be used in Ae. albopictus and that it can be adopted as an efficient tool for genome‐scale analysis and biological study.

C-Type Lectins Link Immunological and Reproductive Processes in Aedes aegypti

“…Recent years have seen major breakthroughs in mosquito gene editing techniques, ranging from the initial demonstration of CRISPR/Cas9 in A. aegypti to the knock-out of kynurenine hydroxylase ( kh ) and dopachrome conversion enzyme ( yellow ), thus creating mosquito white eye (loss of pigment) and yellow body mutants, to the establishment of transgenic germline-specific Cas9 A. aegypti founder strains ( Kistler et al., 2015 ; Li et al., 2017 ; Liu et al., 2018 ; Yang et al., 2019 ). Basu et al.…”

The AalNix3&4 isoform is required and sufficient to convert Aedes albopictus females into males

“…The injected plasmid DNA mixture comprised a construct donor plasmid (500 ng/μL) and the phsp-Pbac helper plasmid [ 19 , 27 , 28 ] (300 ng/μL, a kind gift from Anthony A. James) mixed in injection buffer [ 29 ] and co-injected into 0-1h old embryos [ 30 ]. After microinjection, 16-20h old injected egg were heat shocked at 37°C for 1h, and then placed at 28°C, 80% humidity until hatching.…”

Alternative splicing patterns of doublesex reveal a missing link between Nix and doublesex in the sex determination cascade of Aedes albopictus

“…Two‐d‐old female and male mosquitoes were briefly anesthetized using ice and individually maintained on a glass plate over ice during the injection. Approximately 0.5 μ L of plasmid solution (containing 100 ng/ μ L plasmid and 1× injection buffer [Jasinskiene et al ., 2007; Liu et al ., 2019]) was injected laterally into the thorax using 15 μ m needles with a CellTram ® 4r Oil microinjector (Eppendorf, Hamburg, Germany) under a stereomicroscope (piston position was set to seven–nine to generate optimal positive pressure). After injection, the adult mosquitoes were immediately transferred to small plastic cups (900 mL, 11 cm diameter) and provided a 10% glucose solution.…”