Next-generation gene drive for population modification of the malaria vector mosquito, Anopheles gambiae

Abstract

A Cas9/guide RNA-based gene drive strain, AgNosCd-1, was developed to deliver antiparasite effector molecules to the malaria vector mosquito, Anopheles gambiae The drive system targets the cardinal gene ortholog producing a red-eye phenotype. Drive can achieve 98 to 100% in both sexes and full introduction was observed in small cage trials within 6 to 10 generations following a single release of gene-drive males. No genetic load resulting from the integrated transgenes impaired drive performance in the trials. Potential drive-resistant target-site alleles arise at a frequency <0.1, and five of the most prevalent polymorphisms in the guide RNA target site in collections of colonized and wild-derived African mosquitoes do not prevent cleavage in vitro by the Cas9/guide RNA complex. Only one predicted off-target site is cleavable in vitro, with negligible deletions observed in vivo. AgNosCd-1 meets key performance criteria of a target product profile and can be a valuable component of a field-ready strain for mosquito population modification to control malaria transmission.

Keywords: cage trials; guide RNA polymorphisms; load; nontarget; off-target.

Fig. 1.

Agcd gene, pCO37 gene-drive construct, and resulting phenotypes. (A) Agcd gene: maroon blocks, exons (E1-4); empty blocks, 3′- and 5′-untranslated regions (UTR); thick black line, introns and intergenic DNA. pCO37 plasmid: maroon blocks, homology arms from the Agcd gene; blue blocks, dominant marker gene components (3XP3 and CFP); tan blocks, drive components (nanos promoter and SpCas9 protein-encoding sequences); green blocks, guide RNA components (U6 promoter and gRNA sequence); dark gray block, φC31 attP “docking” site. Genes and features of pCO37 are not to scale and approximate sizes of components in kilobases are listed in the Materials and Methods. Recombination resulting from HDR initiated at the SpCaS9/gRNA-mediated cut site (broken-line arrow) occurring within the pink-shaded regions results in integration of the gene-drive construct. (B) CFP⁺ (blue arrow) and homozygous Agcd-mutant (red arrow) phenotypes in larvae and (C) Agcd-mutant (red arrow) phenotype in pupae. (D) Homozygous Agcd mutant phenotype “red eye” (red arrows) in adults. Approximate image magnifications for B, C, and D are 20, 10, and 20×, respectively.

Fig. 2.

Drive dynamics and population sizes of AgNosCd-1 mosquitoes in discrete, nonoverlapping generations based on small cage trials with 1:1, 1:3, and 1:9 gene-drive to WT males releases. Small cages (5,000 cm³) (A-1, A-2, A-3, B-1, B-2, B-3, C-1, C-2, and C-3) were seeded with 150 WT An. gambiae adult females and 148 to 150 AgNosCd-1 and WT males in ratios of 1:1, 1:3, and 1:9. The resulting next-generation progeny were scored as CFP⁺ (carrying the AgNosCd-1 gene drive cassette) or WT. Percentages of CFP⁺ mosquitoes (y axis) in the total population of each cage were scored at each generation (x axis) (Left). Total population size (y axis) in each replica cage at each generation (x axis) (Right).

Fig. 3.

Distribution of mosquito phenotypes in small cage trials. Relative abundance in percentages (y axis) of eye color and CFP phenotypes of ∼600 randomly selected pupae per generation (x axis) in each cage (A-1, A-2, A-3, B-1, B-2, B-3, C-1, C-2, and C-3). Key: blue, CFP⁺ and WT eye color; salmon, CFP⁺ and cardinal Cd (red) eye color; green, CFP⁺ and tear (mosaic) phenotypes; gray, CFP⁻ and WT eye color; black, CFP⁻ and cardinal.

Fig. 4.

Agcd SNP analysis in wild mosquito populations derived from Africa and SpCas9/gRNA-directed cleavage analysis in vitro. (A) Chromosome coordinates, nucleotide change from the reference allele, nucleotide sequence (5′-3′ orientation) with SNPs in bold red, number of samples heterozygous/homozygous for the SNP, and SNP frequency and sample source. The laboratory variant is from the WT G3 colony. (B) In vitro cleavage assay testing the efficiency of SpCas9/gRNA cleavage of target site SNP variants found in populations from Africa. Plasmids, pCO-57, pCO-58, pCO-59, pCO-60, pCO-61, and pCO-62, containing control (C) WT laboratory colony and variant gRNA sites 1 to 5, respectively, were linearized and used in reactions with SpCas9 and the gRNA. Products at 2,549 and 1,460 bp (large solid blue arrows) are indicative of SpCasp9/gRNA-directed cleavage (uncleaved plasmids are indicated by the large white arrow). Small white arrows show cleavage products in variant 1. M, molecular weight markers.