Figure 1. CRISPR/Cas9-mediated disruption of dsx or ix affects female mosquito morphology and fecundity.

A Schematic maps of gRNA genetic constructs. The gRNA^dsx and gRNA^ix constructs harbor a 3xP3-tdTomato marker and multiple gRNAs guiding Cas9 to the female-specific doublesex (dsx) gene or the female-active intersex (ix) gene, respectively. B A schematic of the genetic cross between the homozygous Cas9 and hemizygous gRNA^dsx/+ or gRNA^ix/+ mosquitoes to generate trans-hemizygous F₁ females (♀’s). Reciprocal genetic crosses were established and two types of trans-hemizygous F₁ ♀’s were generated: gRNA/+; ♀Cas9/+ ♀’s inherited a maternal Cas9; and gRNA/+; ♂Cas9/+ ♀’s inherited a paternal Cas9. Then, both trans-hemizygous ♀’s were crossed to wildtype (WT) males (♂’s), and their fecundity was assessed. C A comparison of the fecundity and male ratio of trans-hemizygous, hemizygous Cas9 or gRNA ♀’s to those of WT ♀’s. The bar plot shows means and one standard deviation (±SD). The data presents one transgenic strain of each construct, gRNA^dsx#1 and gRNA^ix#1, as all strains induced similar results (all data can be found in Supplementary Table 3). D All gRNA^dsx#1/+; Cas9/+ and gRNA^ix#1/+; Cas9/+ trans-hemizygous ♀ mosquitoes exhibited male-specific features (red arrows in D), had reduced fecundity, and were transformed into intersexes (⚥’s). Statistical significance of mean differences was estimated using a two-sided Student’s t test with equal variance (ns: p ≥ 0.05, *p < 0.05, **p < 0.01, and ****p < 0.001). Source data is provided as a Source Data File.