Engineered promoter system enables high-efficiency transgenic CRISPR editing in Malaria transmitting mosquito Anopheles sinensis.

The binary CRISPR/Cas9 system deployed through crosses of transgenic lines facilitates efficient mutagenesis, but its application in non-model insects remains limited by the scarcity of validated species-specific regulatory elements. In the malaria vector Anopheles sinensis, we screened three germline-biased promoters ( Asvasa2, Aszpg, Asnanos) for Cas9 expression, and found that Asvasa2 drove the highest editing efficiency with respect to target site mutagenesis. For gRNA transcription, comparative analysis identified AsU6-1 as the most active of four endogenous U6 promoters. Crossing stable transgenic lines harboring these components yielded F 1 progeny with complete germline editing penetrance at the Aswhite locus, a phenotype inherited in the F 2 generation. Quantitative sequencing of F 1 ovaries confirmed near-saturation (>99%) targeted mutagenesis using the optimal Asvasa2/ AsU6-1 combination, whereas alternative promoters showed markedly lower mutagenesis efficiency. Functional validation through knockout of Asdsx- F, a key sex differentiation regulator, efficiently induced complete female-to-male sexual reversal and sterility. This study provides a foundational genetic toolkit for genome engineering in this vector species, as well as an effective reference for binary transgenic manipulation in non-model insects.