Gene-edited mosquitoes slash malaria spread 93% using self-propagating shield

Outsmarting insecticide resistance: How one genetic edit in mosquitoes self-propagates across generations, cutting malaria transmission to near zero without harming survival. 

Anopheles mosquito larva – Study: Driving a protective allele of the mosquito FREP1 gene to combat malaria. Image Credit: Sinhyu Photographer / Shutterstock

In a recent study published in the journal Nature, a group of researchers investigated whether the fibrinogen-related protein 1 (FREP1) glutamine 224 (Q224) allele renders Anopheles stephensi mosquitoes refractory to Plasmodium infection, quantified the fitness costs associated with this allele, and evaluated a linked allelic drive system to spread this protective variant through populations.

Background

About 600,000 people died of malaria in 2023, mostly children in sub-Saharan Africa and South Asia. Traditional tools, such as insecticide-treated bed nets, indoor residual spraying, and antimalarial drugs, are losing ground to insecticide-resistant mosquitoes and drug-resistant parasites. Gene-drive technologies that spread beneficial alleles through mosquito populations offer a complementary and durable solution. The mosquito FREP1 helps parasites cross the midgut, and a naturally occurring Q224 appears protective without harming mosquito biology. Testing and safely driving such endogenous alleles could reduce transmission while preserving fitness. Further research is needed to validate efficacy, fitness, spread, and containment.

About the study

Using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9), the team generated congenic Anopheles stephensi mosquito strains that differed only at amino acid 224 of FREP1: the wild type (WT) leucine (L224) and the putatively protective glutamine (Q224). A guide ribonucleic acid (gRNA) targeted an intronic site 126 base pairs (bp) upstream of the codon, enabling homology-directed repair (HDR) to install Q224 together with green fluorescent protein (GFP) or red fluorescent protein (RFP) markers. Fitness was assessed by wing length, fecundity, egg hatch rate, pupation, emergence, and adult longevity in Kaplan-Meier survivorship assays.

Vector competence was measured by standard membrane feeding with Plasmodium falciparum (a human malaria parasite) at low and high gametocytemia, and by mouse feeding with Plasmodium berghei (a rodent malaria parasite), which quantified oocysts and salivary gland sporozoites. A linked allelic-drive cassette carrying gRNA L224 was combined with vasa Cas9 to bias inheritance, and multi-generational cage trials (10 generations) tracked marker frequencies. Receiver alleles were genotyped using polymerase chain reaction (PCR), Sanger sequencing, and next-generation sequencing (NGS) to estimate the outcomes of double-strand break (DSB) repair, including non-homologous end joining (NHEJ). Bayesian mathematical modeling inferred conversion rates, fitness costs, and dynamics under laboratory, freely mating cage conditions.

Study results

Across extensive fitness assays, the FREP1Q224 allele imposed negligible overall fitness costs. Wing length, fecundity, egg hatch, pupation, and adult emergence were indistinguishable from the WT leucine 224 (FREP1L224) controls, with only minor, inconsistent differences in male body size and lifespan that did not change competitive performance in multi-generational cages. Virgin females carrying FREP1Q224 lived as long as controls, and blood-fed females showed a slight reduction in longevity compared to virgins, but modest longevity differences in males or blood-fed females did not shift allele frequencies over time.

Infection experiments revealed strong protection in homozygous mosquitoes. At low Plasmodium falciparum gametocytemia (0.08%), infection prevalence dropped from roughly 80% in vasa Cas9 and FREP1L224 controls to about 30% in FREP1Q224 mosquitoes, while the median oocyst number fell from three to zero. Sporozoite burdens in salivary glands plummeted approximately fivefold, from medians above 4,000 to zero. Even at higher gametocytemia (0.15%), the median number of oocysts declined from roughly 32 per midgut to fewer than 10, and sporozoites were sharply reduced. Against Plasmodium berghei, a divergent rodent parasite, FREP1Q224 also lowered median oocyst counts (43 to 25) and sporozoite numbers (approximately 19,000 to 11,000). However, oocyst prevalence did not decline significantly, likely due to the unnatural pairing of mosquitoes and parasites, confirming broad-spectrum refractoriness. Resistance required homozygosity: heterozygotes (FREP1L224/FREP1Q224) were not significantly protected.

The linked allelic drive worked efficiently. In pair mating tests, guide ribonucleic acid L224 plus Cas9 converted 50 to 86% of receiver FREP1L224 alleles to FREP1Q224 by HDR, with higher conversion when Cas9 was maternally provided, producing an overall protective allele frequency of up to 93% in the second generation. Non-homologous end joining mutations were modest (0–12%) and often appeared deleterious. In freely mating population cages seeded at a 1:3 donor:receiver allelic ratio, the frequency of the protective allele rose from 25 percent to above 90% within 10 generations. The fraction of NHEJ alleles declined from 5.4% to less than 0.5%, consistent with fitness costs associated with loss-of-function variants of fibrinogen-related protein 1.

Bayesian modeling supported a synthesis of high allelic conversion, low rates of functional resistance alleles, and lethal sterile mosaicism, where WT homozygotes exposed to maternally deposited Cas9-gRNA complexes suffer severe fitness penalties due to somatic mutation of both FREP1 alleles, as drivers of the observed super-Mendelian spread. Finally, mosquitoes sampled from late-generation cages showed near-complete suppression of Plasmodium falciparum oocysts (median zero to 5.5) at low gametocytemia, confirming that the driven population had become largely transmission refractory. Notably, the protective allele maintained parity in head-to-head competition with congenic chromosomes, reinforcing that its spread reflects drive, not hidden fitness advantages or other ecological costs.

Conclusions

To summarize, this study demonstrates that replacing a single amino acid in FREP1 and biasing its inheritance through a linked allelic drive can render Anopheles stephensi largely refractory to both human and rodent malaria parasites, without incurring substantial fitness penalties. Because the protective variant preserves normal mosquito biology, it offers a realistic, population-friendly path to lower transmission, complementing bed nets, indoor residual spraying, and antimalarial drugs now threatened by resistance. The same framework could convert insecticide resistance alleles back to sensitivity or deploy other protective host variants. Rigorous ecological, ethical, and governance frameworks, as well as confinement strategies, will be essential before any deployment.