Fig. 1: Double-tap trans-complementing gene-drive (DT-tGD) experimental setup and inheritance analysis.

a Schematic of the DT-tGD arrangement in which the Cas9 and gRNA elements are kept as two separate transgenic lines; gRNA-1 and gRNA-2 target the loci at which the Cas9 and gRNA elements are inserted, respectively. When crossed, Cas9 combines with gRNA-1 and gRNA-2 to generate double-strand breaks at each of the wild-type alleles. Repair by end-joining (EJ) pathways rather than homology-directed repair (HDR) would ordinarily halt gene-drive spread. Upon generation of a predicted resistance allele, Cas9 together with double-tap gRNAs gRNA-1b and gRNA-2b can regenerate double-strand breaks at these loci, providing a second chance for the drive elements to be copied by HDR. b gRNAs used in this system. The y1-gRNA and w2-gRNA target the wild-type yellow and white loci, respectively. The y1b-gRNA and w2b-gRNA target a single base pair deletion of the most common indel generated at the yellow and white loci, respectively. c Cross scheme used in this experiment. Males carrying the DsRed-marked Cas9 transgene inserted at the yellow locus are crossed to virgin females carrying the GFP-marked gRNA element inserted at the white locus. Trans-heterozygous virgin F₁ females are single-pair crossed to wild-type males, and the resulting progeny are scored for green and red fluorescence as markers of transgene inheritance. The dark gray half-arrows represent the male Y chromosome. d Transgenic fly lines used in this experiment. vasa-driven Cas9 is marked with DsRed and inserted in the yellow locus. Various gRNA combinations, in which each gRNA is driven by a U6 promoter, are marked with EGFP and inserted in the white locus. e Single female germline inheritance rates as measured by fluorescence phenotypes detected in the F₂ progeny. Black bars represent the average inheritance rates, and blue shaded boxes indicate the deviation from the normally expected 50% Mendelian inheritance. Pie charts represent the percentage of crosses that resulted in 100% inheritance of that transgene.