Fig. 2: Conversion of the insecticide resistant para^L1014F allele to para^WT.

A y^<CC|pF|> allelic-drive element carrying a DsRed marker and two gRNAs: (1) gRNA-y (brown), which sustains copying of the drive element; and (2) gRNA-F (orange), which targets the insecticide resistant, gRNA-sensitive para^L1014F allele (scissors icon) to drive super-Mendelian inheritance of the insecticide-susceptible, uncleavable para^WT allele (lock icon) when Cas9 is provided in trans. In addition, a mini-white marker (w^+mc, red triangle), about 0.5 cM from para^L1014 is used to track the donor chromosome carrying the para^WT allele. B gRNA sequence targeting para^L1014F and its cut-sensitive TTC codon (coding for phenylalanine = F), and the uncleavable CTT para^WT allele (coding for Leucine = L, marked in red). The cut site is indicated with an arrowhead. C Crossing-scheme used to generate F1 “master females”. The yellow shaded X donor chromosome carries the DsRed-marked drive element (y^<CC|pF|>) and the para^WT allele (labeled L) associated with a white⁺ insertion (+w^+mc), which dominantly confers red eye color in the white⁻ mutant background. The receiver white⁻ X chromosome, which carries the cut-sensitive para^L1014F allele, but lacks the w^+mc insertion (−w^+mc in C), is followed through the white⁻ eye color phenotype. A GFP-marked transgene expressing Cas9 (vasaCas9) on the third chromosome is depicted in green and wild-type (+) chromosomes in light gray. Red and orange arrowheads indicate copying of the drive element and the para^WT allele, respectively. D Percent transmission of the drive in the presence (DR, green circles) or absence (gray circles) of Cas9 in females and males. Values indicate mean (±s.e.m) percent transmission for each genotype. Data analyzed using one-way ANOVA followed by Sidak’s multiple comparison tests. Chi-square test for DsRed proportions was also performed in presence and absence of Cas9. p < 0.0001 was seen in both females and males. E Percent survival to 50 ppm DDT in male receiver populations in the presence (green circles) or absence (gray circles) of Cas9. F Percentage conversion at 1014 F locus for receiver chromosomes (selected for w⁻ eye phenotype) was determined based on the proportion of individual F2 male progeny that carried one or the other allele by sequencing of PCR amplified DNA. These F2 males were collected from F1 master females (y^<CC|pF|> drive, w^+mc, para^WT/para^L1014F; Cas9/+ ♀ X w⁻ ♂). E, F Means ± s.e.m are plotted. Data were analyzed by Mann–Whitney test^. *p < 0.033, **p < 0.0021, ***p < 0.0002, ****p < 0.0001, ns not significant.