Lamdan, L. B.; Popovsky-Sarid, S.; Kolley, E. S.; Sarig, A.; Haber, D. A.; Yonah, E. S.; Marois, E.; Davranoglou, L. R.; Arien, Y.; Papathanos, P. A.

Sex-ratio distortion systems are promising genetic tools for mosquito population control. Two strategies have been proposed: prezygotic elimination of X-bearing sperm by X-shredding, which can drive invasive Y-chromosome transmission when sex distorters are Y-linked, and postzygotic daughter killing through disruption of X-linked haploinsufficient genes, a self-limiting approach known as X-poisoning. Previous attempts to implement X-poisoning in the malaria mosquito Anopheles gambiae unexpectedly produced prezygotic distortion, with sex bias arising from loss of X-bearing sperm rather than daughter lethality. Here we use a split CRISPR-Cas9 system to systematically compare sex-ratio distortion outcomes across germline Cas9 drivers and X-linked target genes. Meiotic X-chromosome targeting induced preferential Y-chromosome transmission regardless of target identity, function, or number of sgRNA target sites. In contrast, shifting Cas9 expression to earlier spermatogenic stages altered outcomes dramatically: targeting X-linked ribosomal protein genes caused severe developmental or reproductive toxicity, whereas targeting the haplolethal muscle gene wupA produced daughter-specific post-embryonic lethality, with the majority of surviving females emerging flightless. Tracking offspring using a Y-linked fluorescent marker confirmed that sex chromosome segregation remained unbiased, with female mortality accumulating progressively from the first larval instar, reaching near-complete lethality by adulthood. These results demonstrate that the timing of Cas9 expression during spermatogenesis, rather than target gene identity alone, determines the outcome of X-chromosome targeting in malaria mosquitoes, and establish the conditions required for genuine X-poisoning. Identification of wupA as an effective X-poisoning target provides a solid foundation for the future development of self-limiting Y-linked sex-ratio distortion systems for malaria vector control.