Impact of naturally occurring target-site polymorphisms on an autonomous Cas9/guide RNA-based gene drive system for...

Malaria remains a significant global health challenge with an estimated 282 million cases reported in 2024. CRISPR/Cas9-based gene-drive systems have emerged as promising tools to block Plasmodium transmission by mosquito vectors. The TP13 drive system targets the Anopheles gambiae cardinal (Agcd) gene and carries 2 engineered monoclonal antibodies to achieve rapid population modification to prevent parasite transmission. Previous cage trials demonstrated complete drive introduction in 3 to 6 generations and supported modeling predicting a potential >90% reduction in malaria incidence under optimal conditions. However, naturally-occurring genetic polymorphisms in wild mosquito populations, particularly single-nucleotide polymorphisms (SNPs) within Cas9/guide RNA target sites, pose a potential barrier to drive efficiency. High genetic diversity in An. gambiae results in drive-system target-site variants, including an A→T transversion in the Agcd gene, which occurs at high frequencies in African populations and could affect TP13 drive dynamics. The impact of this and other SNPs on TP13 performance were assessed by establishing 3 An. gambiae Ndokayo lines, 1 with the wild-type Agcd and 2 with homozygous SNP haplotypes. We evaluated drive conversion rates in vivo, population dynamics in cage trials, fitness costs, and parasite suppression efficacy. No negative effects on drive performance and parasite suppression were observed. The results provide insights into the influence of naturally-occurring polymorphisms on gene drive propagation, informing safety, efficacy, and target product profile requirements for advancing gene-drive mosquitoes toward field trials.