Mosquito sex separation using complementation of selectable traits and engineered neo-sex chromosomes

Effective sex separation remains a critical challenge for mosquito genetic control. Genetic sexing strains (GSS) address this by linking maleness with selectable traits, enabling efficient female removal. Here, we present a versatile platform for GSS development in the invasive Aedes albopictus mosquito that integrates CRISPR-engineered selectable phenotypes with sex conversion via nix, the male-determining factor. As a proof-of-concept, we disrupt the yellow pigmentation gene and restore its function in males using nix-containing transgenes, producing a stable strain with yellow females and dark males. Beyond serving as a vivid marker, yellow confers added advantages: GSS females pupate later than wild females, enhancing protandry-based sorting, and lay desiccation-sensitive eggs, lowering accidental female release risk. The strain is compatible with size-based separation, improving sexing accuracy through the integration of natural and engineered dimorphisms. To our knowledge, this represents the first engineered sex-linked selectable trait in mosquitoes based on endogenous genes, establishing a foundation for scalable GSS development.