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

(A) Schematic map 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 wild-type (WT) males (♂’s), and their fecundity was assessed. 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) over triple biological replicates (n = 3). The data presentsone 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 file 1c). (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 are provided in Supplementary file 1.

Figure 1—figure supplement 1. Changes in morphological structures induced by disruption of targeted genes.

External and internal structures were measured in mosquitoes of each genotype and sex. The trans-hemizygous mosquitoes harboring the maternal Cas9 (Cas9^♀) were used for all measurements. The plot shows means ± standard deviation (SD) over 15 mosquitoes (n = 15) for each morphological structure. All data can be found in Supplementary file 1b. 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 are provided in Supplementary file 1.

Figure 1—figure supplement 2. Induced mutations at the targeted sequences in dsx and ix.

The gene sequences targeted by gRNAs were Polymerase Chain Reaction (PCR) amplified from precision-guided sterile insect technique (pgSIT) mosquitoes and Sanger sequenced. Good quality of Sanger read chromatogram indicates the presence of a single consistent temple sequence (no cleave) at a targeted region, for example gRNA^dsx#3 (A) or gRNA^is#2 (B). An active CRISPR/gRNA-mediated cleavage at a particular DNA target results in re-ligation of cut fragments leading to the origin of diverse mutation-bearing alleles. These induced mutations are localized around the gRNA cleavage site and result in the drop of Sanger read quality (cleavage) as indicated for gRNA^dsx#1, gRNA^dsx#2, gRNA^dsx#4, (A) and gRNA^is#1 (B).

Figure 1—figure supplement 3. Ae. aegypti dsx or ix disruption does not result in significant alterations in ear anatomy, wing beat frequency (WBF), and male phonotactic behaviors.

Immunohistochemistry and microanatomic comparison of mosquito Johnston’s Orgran (cup-like structure) with chordotonal neurons responsible for auditory transduction in females (♀’s, A) or males (♂’s, B) among different genotypes. Female JO’s contained far fewer neurons than males across all genotypes, and distributions of synaptic punctate assessed by Anti-Synapsin (3C11, in green) were restricted to the somata, as opposed to the male-type distribution observable in the gap between the cilia and somata tracked by anti-horseradish peroxidase (HRP, in red). WBFs are different between Ae. aegypti ♀’s and ♂’s, and are shown on separate plots, (C) and (D), respectively. Each point represents the estimated frequency of a single fly-by (aka. the frequency of sound produced by one event of a mosquito flying past the microphone). WBF box plots indicate the median (middle bar), and 25% and 75% quartiles (lower/upper boundaries of box). The average ♀ WBF is 468 Hz. No significant differences were observed between ♀ groups, or between ♂ groups, regardless of the genotypes tested (analysis of variance [ANOVA] on ranks, p > 0.05). The male phonotaxis assay was conducted, in which individual flying ♂’s showed attractive or no response (scored as 1 or 0, respectively) toward the female-specific WBF produced for 30 s by a speaker (E). Three batches (aka. 3 replicate groups) of at least 10 ♂’s were examined for each genotype. ♂’s across all genotypes exhibited robust responses to sound. The point plot shows the mean (middle bar) and one standard deviation (± SD) for the proportion of ♂’s responding to the auditory stimulus (n=3). No significant differences were observed across groups (Chi-squared test, p > 0.05).