By Pooja Toshniwal PahariaReviewed by Lauren HardakerJul 31 2025In a recent study published in Nature, researchers developed a novel genetic approach to combat malaria using a naturally occurring protective allele of the mosquito's fibrinogen-related protein 1 (FREP1) gene. The Q224 allelic variant of FREP1 confers strong resistance to human (P. falciparum) and rodent (P. berghei) malaria parasites in Anopheles stephensi mosquitoes, with a largely fitness-neutral profile.
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However, some modest and sex-specific differences in lifespan were observed. The engineered mosquitoes showed reduced parasite infection without significant fitness costs, making them a promising tool for malaria elimination. The study also introduces a linked allele-gene drive system to replace codon L224 with a malaria parasite-resistant Q224 allele, making mosquito populations resistant to infection.
Malaria is a devastating disease, causing thousands of deaths annually. Efforts to reduce malaria deaths through indoor residual sprays, anti-malarial drugs, and insecticide-treated bed nets are currently used. However, the emergence of drug-resistant parasites and insecticide-resistant mosquito vectors significantly set back malaria control efforts. The lack of efficient approaches necessitates alternate strategies, such as genetically altered mosquitoes.
Current genetic engineering strategies involve suppressing mosquito counts or modifying them to prevent the transmission of parasites. However, these approaches face challenges in functionality, fitness costs, effector expression, and stability. Issues like poor coordination between meal (blood)- induced expression and parasite infection dynamics limit their effectiveness.
Previous studies have reported that the FREP1Q allele prevents parasite infection while maintaining critical physiological functions in the mosquito. However, this protective effect has not undergone rigorous testing in stable mosquito colonies with defined genotypes until now. Correlative genomic evidence for FREP1 equivalents in other Anopheline species is limited.
About The Study
In the present study, researchers investigated the genetic modification of Anopheles stephensi mosquitoes to control malaria.
The team-maintained Anopheles stephensi wild-type (WT) and transgenic vasa-Cas9 mosquito lines under controlled laboratory conditions (27°C, 77% humidity) for over 30 generations. They performed microinjections on pre-blastoderm vasa-Cas9 embryos using donor plasmids.
The researchers inserted a gene cassette with a fluorescent marker (IE1-RFP-SV40 or IE1-eGFP-SV40) into the FREP1 gene’s second intron to generate the FREP1Q224 allelic variant. Replacing the parasite-susceptible L224 codon with resistant Q224 in the donor plasmid created congenic strains, differing in only one amino acid. Fitness cost evaluation parameters for the FREP1Q224 allele included wing length (proxy for body size), fecundity (egg production and hatching rates), and longevity.
Artificial membrane feeding assays revealed the ability of Anopheles stephensi mosquitoes to act as vectors for P. berghei and P. falciparum. The researchers fed mosquitoes 0.08-0.15% P. falciparum gametocyte culture mixed with human blood. For P. berghei, they infected mice with the rodent parasite. They measured the prevalence and intensity (number of oocysts per midgut) of oocyst infection at eight days (P. falciparum) and 12 days (P. berghei) post-infection. They also quantified sporozoite loads in salivary glands after two weeks of infection.
The team crossed transheterozygous F1 mosquitoes with WT A. stephensi and tabulated F2 progeny phenotype and genotype. They assessed three-generation mating to evaluate allelic conversion rates and Sanger sequencing to determine the allelic conversion frequency.
Next-Generation Sequencing (NGS) genotyped mosquitoes and inferred their allelic frequencies in multi-generational competition cage trials with transheterozygous mosquitoes seeded in cages at a 1:1 allelic frequency. Bayesian mathematical modeling provided additional insight, suggesting that lethal or sterile mosaicism contributed to the rapid spread of the protective allele by eliminating individuals carrying only the susceptible L224 allele and Cas9.
Results
The FREP1Q224 allele conferred strong resistance to P. berghei and P. falciparum malaria parasites. Specifically, mosquitoes with the FREP1Q224 allele showed a significant reduction in P. falciparum oocyst infection prevalence (from 80% in controls to 30% at low gametocytemia) and intensity (median oocysts per midgut decreased from 3 to 0). Similar reductions were observed at high gametocytemia and for P. berghei infection. The findings indicate that the FREP1Q224 allele effectively inhibits parasite development at early pre-oocyst stages.
A crucial finding is that the FREP1Q224 allele is largely fitness-neutral in Anopheles stephensi mosquitoes. Assessing mosquito wing length, fecundity, and longevity revealed minimal and context-specific fitness costs associated with the FREP1Q224 allele compared to wild-type or control strains. Fitness neutrality is vital for the successful deployment and persistence of gene-edited mosquitoes in wild populations.
The novel linked allelic-drive system (replacing L224 with Q224) efficiently propagated the protective FREP1Q224 allele. In multi-generational cage experiments, the FREP1Q224 allele's frequency rapidly increased from the initial 25% seeding percentage to over 90% within 10 generations, demonstrating efficient super-Mendelian transmission.
The system also showed a low rate of non-homologous end-joining (NHEJ) alleles, which progressively decreased over generations, indicating that the drive system is robust and does not produce a significant fraction of interfering alleles. The FREP1Q224 allele provided broad-spectrum resistance, effective against different malaria parasite species, highlighting its potential for widespread application in malaria control.
The study findings demonstrate the feasibility and effectiveness of using a gene-edited FREP1Q224 allele and an efficient gene drive to create malaria-resistant mosquito populations. This strategy offers a powerful new approach for malaria control. Notably, the drive was more effective when the donor and target alleles shared extensive intronic homology, suggesting that future optimization of drive design may enhance conversion rates in field-relevant populations.
Mathematical modeling also highlighted that moderate gene conversion rates, low levels of NHEJ formation, and reduced fitness of unconverted L224 alleles in the presence of Cas9 drove the system’s success.
Journal Reference
Li, Z., et al. (2025). Driving a protective allele of the mosquito FREP1 gene to combat malaria. Nature. DOI: 10.1038/s41586-025-09283-6 https://www.nature.com/articles/s41586-025-09283-6
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