Figure 3. Timing and expression of drive components affect cleavage and transmission rates.

We bidirectionally crossed trans-heterozygous mosquitoes that contained both components of a gene drive: the gene drive element (GDe) and the Cas9 transgene (w^GDe/w+; Cas9/+). (Figure 2A). (A–C) Four w^GDe/w+ lines, each with a different gRNA^w U6 promoter, were crossed to the exu-Cas9 strain to compare cleavage and homing activity. (A) Maternally deposited Cas9 protein induced white cleavage in G₁ trans-heterozygotes harboring w^U6a-GDe, w^U6b-GDe, and w^U6c-GDe but not w^U6d-GDe. (B) In comparison to w^GDe/w+, trans-heterozygous w^GDe/w+; exu-Cas9/+ females, (C) but not males, exhibited super-Mendelian transmission of w^GDe. The w^U6b-GDe/w+; exu-Cas9/+ females transmitted w^U6b-GDe to 70.9 ± 7.8% of G₂ progeny. (D–F) Five lines expressing Cas9 from different promoters were crossed to w^U6b-GDe/w+. (D) Maternally deposited Cas9 resulted in white cleavage in G₁ trans-heterozygotes. (E) Three out of five tested Cas9 lines, exu-Cas9, nup50-Cas9, and ubiq-Cas9, induced super-Mendelian transmission of w^GDe by trans-heterozygous females. The w^U6b-GDe/w+; nup50-Cas9/+ females transmitted w^U6b-GDe to 80.5 ± 5.0% of G₂ progeny. (F) All trans-heterozygous males transmitted w^GDe following a regular Mendelian inheritance except for the w^U6b-GDe/w+; nup50-Cas9/+ males that induced white cleavage in 51.0 ± 3.9% and transmitted the w^U6b-GDe allele to 66.9 ± 5.4% of G₂ progeny. Point plots show the average ± standard deviation (SD) over 20 data points. Grey dotted line indicates standard Mendelian inheritance rates. Statistical significance was estimated using an equal variance t-test. (P ≥ 0.05^ns, p<0.05*, p<0.01**, and p<0.001***).

Figure 3—figure supplement 1. Split-drive over multiple generations.

Frequencies of white cleavage and w^U6b-GDe transmission were plotted over multiple generations. The w^U6b-GDe/w+; exu-Cas9/+ and w^U6b-GDe/w+; nup50-Cas9/+ trans-heterozygous females, or males were outcrossed to wt each generation and both transmission and cleavage rates were scored. Average transmission rates at consecutive generations were compared to the corresponding rates at G₂. Point plots show the average ± SD over 20 data points. Statistical significance was estimated using a t test with equal variance. (P ≥ 0.05^ns, p<0.05*, and p<0.001***).

Figure 3—figure supplement 2. Sequences of de novo resistance alleles at white locus (w^R).

Mosquitoes carrying resistance alleles (w^R) were identified among progeny of genetic crosses between w^U6b-gRNA/w+; nup50-Cas9/+ trans-heterozygous mosquitoes with wt (Supplementary file 6) and were further crossed to the Ae. aegypti loss-of-function w^∆19/w^∆19 line, to place novel knockout alleles (w^R) into the recessive genetic background. To sample w^R alleles from w^∆19/w^R heterozygous mosquitoes, PCR amplicons amplified from an individual mosquitoes were cloned into a plasmid, and a seven clones were Sanger sequenced in both directions and aligned against the w+ and w^∆19 alleles in SnapGene 4.2. Genders and genotypes of analyzed mosquitoes as well as the generation when they were recovered are provided on the left side. Numbers of bases deleted is indicated below the corresponding deletion. Notably, while the deletions of 3 and 18 bases re-established the reading frame for white gene, they still displayed the loss-of-function phenotype.