A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes

TLDR

The doublesex gene in Anopheles gambiae produces male and female transcripts that control sex differentiation, with the female transcript containing a highly conserved exon 5 shared across Anopheles species. A CRISPR–Cas9 gene drive targeting this conserved exon 5 of doublesex caused rapid spread to 100 % prevalence in 7–11 generations, produced intersex sterile females, and led to complete population collapse in cages without selecting for resistant alleles.

Abstract

Complete population collapse of malaria vector Anopheles gambiae in cages is achieved using a gene drive that targets doublesex. In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.