Fig. 1. The Reckh gene drive.

a Swap strategy for Cas9/gRNA-mediated cassette exchange. Two plasmid-encoded gRNAs (top) guide cleavage in the genome of the white-eyed nRec mosquito line (kh^nRec–)⁸ (middle), leading to the excision of a fragment including the DsRed eye (3xP3) marker and the two antimalarial effectors m2A10 and m1C3¹¹. The HDR template plasmid (bottom) carries homology arms flanking either cut site, promoting the insertion of a GFP-marked donor template that carries a recoded portion of the kh gene followed by the 3′-end sequence of the An. gambiae kh gene including the 3′UTR (A.gam.3′) to minimize homology. b The insertion of this unit restores kh gene function while creating a sequence (kh^Rec+) that is uncleavable by the endogenous drive components. c The Reckh gene-drive includes an An. stephensi codon-optimized Cas9 driven by the germline-specific vasa promoter from An. stephensi and a gRNA (gRNA-kh2) directed to the fifth exon of the unmodified kh⁺ gene (top) regulated by the ubiquitous promoter of the An. stephensi U6A gene⁸. The cut in the kh gene of the Reckh mosquito germline can be repaired by drive integration via HDR (homology-directed repair) or by the less desirable EJ (end-joining) pathway (bottom). HDR results in the integration of the drive cassette that maintains kh gene function at the integration site (kh^Rec+), while EJ usually causes the formation of loss-of-function alleles (kh⁻). When function is lost in both copies of the gene, individuals with white eyes are produced. kh, kynurenine hydroxylase gene; attP, φC31 recombination site; U6A, RNA polymerase-III promoter; gRNA, guide RNAs; Cas9, Cas9 open reading frame; vasa, vasa promoter; 3xP3, eye-marker promoter; GFP, green fluorescent protein; dominant marker gene. The horizontal dimension of the mosquito heads at the eyes in the images is ~1 mm.