Figure 2. Assessing super-Mendelian inheritance of split drives.

(A) Crossing scheme of the w^GDe and exu-Cas9 parent strains (P) to generate trans-heterozygotes (G₁), and the outcrossing of their progeny to wild-type (wt) mosquitoes (G₂). tdTomato eye and dsRed abdominal expression were the w^GDe and exu-Cas9 transgene inheritance markers, respectively. w^GDe transmission and white loss-of-function mutation rates were estimated among G₂ progeny of trans-heterozygous female and wt male crosses. Super-Mendelian inheritance of w^GDe occurred when the transmission rate of w^GDe in G₂ progeny was >50%, as expected by standard Mendelian inheritance. (B) Examples of G₂ progeny eye phenotypes and corresponding genotypes.

Figure 2—figure supplement 1. Sex biased inheritance of the GDe is due to linkage of white and Nix, a male determining gene in Ae. aegypti.

Schematic of site-specific integration and sex-linked inheritance of Gene Drive element (GDe) (A). To direct site-specific integration of GDe inside white locus (white stripe on chromosome I) via homology directed repair (HDR), wild type (wt) Ae. aegypti embryos were injected with a Cas9/gRNA^w complex and a plasmid carrying GDe flanked by homology regions complementary to left and right genomic regions at the white cut site. GDe contains two genes: U6-gRNA^w to direct a site-specific cleavage and the 3xP3-tdTomato transgenesis marker (red stripe). Female mosquitoes carrying w^GDe/w+ passed w^GDe randomly to both genders, while transgenic males transmitted w^GDe either to females (type I) or males (type II) nearly exclusively. Close genetic linkage of white and Nix genes causes tight sex-linked inheritance of w^GDe via male founders (B). Nix is located near the centromere of chromosome I (Dudchenko et al., 2017; Matthews et al., 2018) and consequently, type I male (♂) founders have w^GDe inserted on the chromosome copy without Nix and therefore pass w^GDe exclusively to female progeny. On the other hand, type II male founders have w^GDe integrated on the chromosome with Nix, and thus they transfer w^GDe and Nix together exclusively to male progeny (box). Bars show average ± SD estimated for 20 data points. Statistical significance between gender frequencies was estimated by an equal variance t test. (P ≥ 0.05^ns and p<0.001***).

Figure 2—figure supplement 2. Site-specific integration of Gene Drive element (GDe).

Schematic showing positions of Left and Right Homology Arms (LHA and RHA) of GDe relative to genomic white sequence. The cut site is presented over the genetic structure of white, with darker gray boxes representing exonic coding sequences. GDe constructs were integrated at the cut site via homology directed repair (HDR) relying on the construct’s LHA and RHA to white. Arrows depict primers (AE20, AE21, AE22 and AE23) used for PCR and their location relative to genomic and constructs sequences. Two gel images showing specific PCR amplicons for each side of integration (B). The same DNA samples, listed in the box to the left, were used to PCR of both fragments. To confirm PCR specificity, amplicons were Sanger sequenced from both ends.