A Modular Donor Plasmid System for Efficient CRISPR-Based Genome Engineering in Aedes aegypti

Abstract

Mosquito host-seeking behavior depends on olfactory detection of human odor, yet functional analysis of individual receptors is limited by the technical difficulty of assembling multi-component donor plasmids for genome editing in Aedes aegypti. In this study, a modular HDR donor plasmid system was developed using the pJAT77 (Atalanta) backbone, incorporating a species-specific U6-driven guide RNA cassette and Gateway recombination for efficient insertion of locus-specific sequences. The system was validated through assembly of a donor construct targeting the IR75d locus, with sequence-confirmed integration of homology arms and reporter elements. Compared to traditional cloning approaches, this strategy reduced hands-on time and increased consistency across assemblies. By separating backbone preparation from locus-specific design, the system enables rapid and reproducible generation of donor constructs across multiple genomic targets. This platform provides a scalable method for genetic interrogation of olfactory receptors and supports efforts to link receptor function to mosquito host-seeking behavior.