Abstract
The spotted-wing drosophila (Drosophila suzukii) is an invasive agricultural pest responsible for severe economic losses in soft-skinned fruit production worldwide. Conventional management strategies rely heavily on synthetic chemical insecticides, which present severe ecological liabilities, promote chemical resistance, and pose non-target toxicity risks. In this study, we present the design, biochemical validation, and population-level evaluation of a novel multiplexed CRISPR/Cas9-based homing gene drive engineered for targeted population suppression of D. suzukii. The system targets an ultra-conserved functional exon of the female-specific sex determination gene doublesex (dsx-F), driven by the germline-specific nanos promoter and paired with a multiplexed dual-guide RNA cassette targeting conserved cleavage sites to suppress the formation of functional resistance alleles. Embryonic microinjections and subsequent lineage crosses demonstrated a mean homing efficiency of 96.8% and super-Mendelian transmission in heterozygous drive-carrying females. Disruption of the dsx-F locus resulted in complete sterility and intersex morphological phenotypes in homozygous drive females, while heterozygous females and drive-carrying males retained substantial reproductive fitness. In continuous laboratory cage trials, an initial introduction of 20% drive-heterozygous males led to complete population collapse within 8 to 11 generations without the emergence of drive-resistant functional mutants. These biochemical and genetic findings highlight the potential of highly targeted endonuclease-based gene drives as robust, species-specific, and environmentally sustainable tools for agricultural pest management.
