Malaria is a major global health issue, causing over 600,000 deaths annually. Current control strategies, like antimalarial drugs and insecticide-treated nets, are facing more challenges because of resistance to both Plasmodium parasites and mosquito vectors. A potential alternative is the genetic modification of mosquitoes to inhibit parasite development. The mosquito gene FREP1 plays an important role in allowing malaria parasites to penetrate the midgut epithelium. It is a critical bottleneck in the life cycle of a parasite. Earlier studies indicated that a naturally existing variant, FREP1Q224, can prevent infection. However, conclusive genetic evidence in defined mosquito populations has been lacking. This study aimed to explore whether replacing the susceptible FREP1L224 allele with FREP1Q224 confers resistance to the malaria parasite without compromising mosquito fitness.
Researchers developed Anopheles stephensi strains in which only the FREP1L224 codon was modified using CRISPR-Cas9 genome editing. This involved 2 strains which carry the Q224 variant (FREP1REP-Q and FREP1GEP-Q) and one control strain with the L224 allele (FREP1GFP-L). Genetic modifications were tracked using fluorescent markers. Fitness was assessed through different parameters like fecundity, body size, hatching rate, development, and longevity. Across these metrics, the FREP1Q224 allele revealed low to no fitness cost. While a minor difference was observed specifically in male wing size and reproductive results, this difference did not persist over generations due to random variation. Overall reproductive performance and longevity were comparable between strains, indicating that the Q224 allele is fitness neutral.
Infection resistance was evaluated by both rodent (Plasmodium berghei) and human (Plasmodium falciparum) malaria parasites. In membrane-feeding tests with P. falciparum, FREP1Q224 mosquitoes showed significantly reduced infection intensity and prevalence. Infection prevalence decreased from 80% in controls to 30% in FREP1Q224 mosquitoes at low gametocyte levels (0.08%). Median oocyst counts dropped from 3 to 0. Sporozoite loads in the salivary glands, which indicate potential transmission, were decreased 5-fold or more. Similar decreases in the infection intensity were observed in P. berghei, though changes in prevalence were less pronounced. Heterozygous FREP1L224/Q224 mosquitoes did not exhibit resistance, which indicates that only homozygous Q224 gives protection.
The researchers created a linked allelic drive system to facilitate the spread of the protective allele. This system involved a fluorescent marker, a Q224 mutation, and a guide RNA targeting the susceptible allele. When it is paired with a source of Cas9, this system allows conversion of the L224 allele to Q224 by homology-directed repair. In the lab-based population cage studies, the frequency of the FREP1Q224 allele increased to 90% from 25% in just 10 generations. The rate of unintended mutations from non-homologous end-joining (NHEJ) was low and reduced over time. Mosquitoes from the final generations demonstrated strong resistance to P. falciparum, with median oocyst counts reduced to zero in many instances.
Statistical analyses confirmed the robustness of the results. The Mann–Whitney U-tests revealed a significant reduction in the infection intensity (P < 0.0001), whereas Fisher’s exact tests showed a reduced prevalence. Kaplan–Meier analyses and one-way ANOVA revealed no significant differences in fitness across strains. Bayesian modelling explained the rapid allele spread, which is attributed to low fitness costs, high conversion efficiency, and selective disadvantage of L224 homozygotes exposed to Cas9.
In conclusion, the FREP1Q224 allele efficiently prevents the development of the malaria parasite when homozygous, with minimal fitness cost to mosquitoes. The linked allelic drive system provides an effective method for disseminating the protective allele in the mosquito populations. These results offer a promising genetic approach to decrease malaria transmission and may be applicable to other mosquito species or resistance targets.
Reference: Li Z, Dong Y, You L, et al. Driving a protective allele of the mosquito FREP1 gene to combat malaria. Nature. 2025. doi:10.1038/s41586-025-09283-6
