Mosquito Sex Separation using Complementation of Selectable Traits and Engineered Neo-Sex Chromosomes

Zaada, D. S.; Toren, O.; Krsticevic, F.; Haber, D. A.; Gildman, D.; Galpaz, N.; Haecker, I.; Schetelig, M. F.; Marois, E.; Arien, Y.; Papathanos, P. A.

doi:10.1101/2025.03.30.646185

bioRxiv · 10.1101/2025.03.30.646185

Mosquito Sex Separation using Complementation of Selectable Traits and Engineered Neo-Sex Chromosomes

Zaada, D. S.·Toren, O.·Krsticevic, F.·Haber, D. A.·Gildman, D.·Galpaz, N.·Haecker, I.·Schetelig, M. F.·Marois, E.·Arien, Y.·Papathanos, P. A.

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Abstract

Effective and scalable sex separation remains a critical challenge for mosquito genetic control strategies. Genetic sexing strains (GSS) address this by genetically linking maleness with selectable traits, enabling efficient removal of females before release. Here, we describe a robust platform for the development of GSSs in the invasive Aedes albopictus mosquito by integrating a CRISPR-engineered selectable phenotype with sex conversion via nix, the male-determining factor. As a proof-of-concept, we disrupt the yellow gene to generate a vivid pigmentation marker, then rescue its function in males using nix-containing transgenes, creating a stable strain where all females are yellow and all engineered males are dark. The resulting GSS males are fertile, robust, and despite lacking the ancestral M locus, exhibit gene expression profiles closely resembling wild-type males. We benchmark sex separation based on pigmentation and discover that yellow mutant females exhibit slower larval development, enhancing protandry-based sorting. The GSS strain is compatible with existing size-based sex sorting systems, allowing for improved separation accuracy through the integration of natural and engineered sexually dimorphic traits. Additionally, we find that GSS females lay desiccation-sensitive eggs, reducing the risk of accidental female releases. Our approach is the first to engineer a sex-linked selectable trait by precisely targeting an endogenous gene and restoring its function in males, establishing a versatile platform for GSS development in Aedes mosquitoes.

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Zaada, D. S., Toren, O., Krsticevic, F., Haber, D. A., Gildman, D., Galpaz, N., Haecker, I., Schetelig, M. F., Marois, E., Arien, Y., Papathanos, P. A.. 2025-03-31. Mosquito Sex Separation using Complementation of Selectable Traits and Engineered Neo-Sex Chromosomes. https://doi.org/10.1101/2025.03.30.646185

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