Fig. 2: Development of X-chromosome targeting SRD strains in An. gambiae.

a Relative position of candidate haploinsufficient target genes and the 28S rDNA cluster on the An. gambiae X-chromosome. b Structure of targeted X-linked ribosomal protein genes indicating exons, introns, and coding sequences (CDSs). The location, direction, and sequence of sgRNA target sites are also presented, with PAM sites shown underscored. c Schematic representation of transformation constructs. A total of five distinct constructs were generated, from three different types (single sgRNA X-poisoning, double sgRNA X-poisoning, and X-poisoning/X-shredding). (pBac) piggyBac inverted repeats; (3xP3-DsRed) the 3xP3 promoter driving DsRed fluorescent marker; (β2tub-Cas9) codon-optimized CDS of Cas9 endonuclease under the control of the male germline specific β2tubulin promoter; (U6-sgRNA) sgRNA under the control of the ubiquitous U6 Pol III promoter. d Overview of experimental crosses. Transgenic mosquitoes are indicated with a semi-transparent red circle, indicating 3xP3-DsRed expression. (WT) wild-type e Percentage of F1 adult males within each independent transgenic strain and the wild-type strain (WT). Data are presented as mean values +/-SEM. A total of 9 independent transgenic strains were generated (X-axis), with strains carrying the same transgenic constructs being colored the same. For every transgenic X-targeting strain, both male (solid color) and female (transparent with hatch) crosses are shown. “n” indicates the number of biological replicates used to derive statistics, with the total number of adult F1 individuals that were counted and sexed per cross provided in brackets. Statistical significance was derived from a One-Way ANOVA of the percentage of F1 adult males by strain/cross combination (F(18, 158) = 37.6, p < 0.0001), followed by Tukey-Kramer HSD for comparisons between all pairs (α = 0.05). Different letters indicate statistical difference using Tukey-Kramer HSD. Individual data points represent data from each replicate. Source data are provided as a Source Data file.