The pgSIT cross results in nearly complete female lethality and male sterility.
A Schematic maps of targeted genes (box) and gRNAdsx,ix,βTub construct. Red arrows show relative locations of gRNA target sequences (box). gRNAdsx,ix,βTub harbors a 3xP3-tdTomato marker and six gRNAs to guide the simultaneous CRISPR/Cas9-mediated disruption of dsx, ix, and βTub genes. Violet exon boxes 5a and 5b represent ♀-specific exons. B A schematic of the reciprocal genetic cross between the homozygous Cas9, marked with Opie2-CFP, and homozygous gRNAdsx,ix,βTub to generate the trans-hemizygous F1 (aka. pgSIT) progeny. Relative positions of dsx, ix, and βTub target genes (bar color corresponds to gRNA color), and transgene insertions in the Cas9 (Nup50-Cas9 strain1) and gRNAdsx,ix,βTub#1 strains are indicated in the three pairs of Ae. aegypti chromosomes. To assess the fecundity of generated pgSIT mosquitoes, both trans-hemizygous ♀’s and ♂’s were crossed to the WT ♂’s and ♀’s, respectively. C Comparison of the survival, sex ratio, and fertility of trans-hemizygous, hemizygous Cas9 or gRNAdsx,ix,βTub#1, and WT mosquitoes. The bar plot shows means ± SD over triple biological replicates (n = 3, all data in Supplementary Table 4). D Blood feeding assays using both types of trans-hemizygous intersexes (IZ’s): pgSIT♀Cas9 and pgSIT♂Cas9 IZ’s. To assess blood feeding efficiency, individual mated ♀’s or IZ’s were allowed to blood feed on an anesthetized mouse inside a smaller (24.5 × 24.5 × 24.5 cm) or larger cage (60 × 60 × 60 cm), and we recorded the time: (i) to initiate blood feeding (i.e., time to bite); and (ii) of blood feeding (i.e., feeding time). The plot shows duration means ± SD over 30 ♀’s or IZ’s (n = 30) for each genetic background (Supplemental Table 5). E Flight activity of individual mosquitoes was assessed for 24 hours using vertical Drosophila activity monitoring (DAM, Supplemental Table 6). F Mating assays for fertility of offspring produced via crosses between trans-hemizygous ♂’s that inherited a maternal Cas9 (pgSIT♀Cas9) or paternal Cas9 (pgSIT♂Cas9) and WT ♀’s (Supplemental Table 8). The plot shows fertility means ± SD over three biologically independent groups of 50 WT ♀’s (n = 3) for each experimental condition. Statistical significance of mean differences was estimated using a two-sided Student’s t test with equal variance. (ns: p ≥ 0.05ns *p < 0.05, **p < 0.01, and ***p < 0.001). Source data is provided as a Source Data File.