A male-biased sex-distorter gene drive for the human malaria vector Anopheles gambiae

The study addresses whether coupling a male-biasing sex distorter to a CRISPR-based homing gene drive can rapidly suppress populations of the malaria vector Anopheles gambiae by reducing female numbers and driving the construct through populations. Natural sex-chromosome drivers bias progeny sex ratios and can cause population crashes, but resistance and unknown mechanisms limit their utility. Synthetic X-shredding systems using nucleases such as I-PpoI or CRISPR-Cas9 can bias progeny toward males, yet attempts to create effective Y-linked drives are constrained by meiotic sex chromosome inactivation. Recent gene drives targeting the doublesex (dsx) gene achieved complete population suppression without resistance, suggesting dsx as a robust target. The authors hypothesized that an autosomal sex-distorter gene drive (SDGD) that avoids sex-chromosome transcriptional silencing and combines sex-ratio distortion with homing could accelerate reduction of biting (female) mosquitoes and enhance robustness against resistance, leading to population collapse.

Prior work has characterized natural X and Y drives in insects and their potential for population suppression, alongside observed evolution of suppressors (e.g., Drosophila). Synthetic sex-ratio distorters in Anopheles gambiae using I-PpoI or CRISPR to cleave X-linked rDNA repeats produced ~95% male progeny, though conversion to Y-linked drives has been impeded by meiotic sex chromosome inactivation. CRISPR homing drives targeting female fertility genes and, notably, the dsx gene have achieved rapid spread and suppression in caged populations, with dsx drives showing minimal resistance development due to functional constraint at the target site. Modeling studies have highlighted the potential of homing endonuclease genes and Y-drive systems for vector control but also the risk of resistance and sensitivity to fitness effects. Collectively, these studies motivate combining a sex distorter with a robust homing drive at a constrained locus to enhance efficacy and durability.

• Construct design: Built an autosomal SDGD plasmid combining (i) an X-chromosome shredding endonuclease I-PpoI (W124L variant) fused to eGFP under a male germline beta2-tubulin promoter, (ii) SpCas9 driven by germline promoters (initially vasa, later optimized to zpg), (iii) a U6-driven gRNA targeting female fertility genes or the dsx intron 4–exon 5 boundary, and (iv) a 3xP3::DsRed marker. The sex-distorter and drive components were linked head-to-tail and inserted into target autosomal loci via RMCE.
• Initial targets and assessment: Generated SDGD constructs targeting AGAP011377, AGAP007280, and AGAP005958. Crossed heterozygotes to wild type to quantify inheritance (RFP+), sex ratio, and life-history traits. Identified severe fitness costs (reduced female and male fertility) likely due to vasa-driven Cas9 somatic/maternal activity and high I-PpoI expression, impeding spread in cages.
• Promoter optimization: To minimize ectopic Cas9 activity, replaced vasa with zpg regulatory elements. To reduce I-PpoI expression that caused male sterility, engineered attenuated beta2-tubulin promoters by inserting a 100-bp GC-rich sequence at positions −244, −271, or −355 relative to ATG; selected beta2^244 variant with ~8.1% activity of wild type based on dual-fluorescence reporter assays in testes.
• Final SDGD targeting dsx: Built SDGDdsx with zpg-Cas9, beta2^244–I-PpoI, and a gRNA targeting the dsx intron 4–exon 5 boundary (AGAP004050), a functionally constrained site previously shown to limit resistance.
• Phenotypic assays: Performed single-pair crosses of SDGDdsx heterozygous males or females to wild type; quantified egg and larval counts, hatching rates, inheritance bias (RFP), and sex ratios in progeny. Assessed parental origin effects on fertility.
• Cage trials: Established caged populations of 600 mosquitoes and released SDGDdsx heterozygotes at starting allelic frequencies of 2.5% (male-only release) or 25% (both sexes), in duplicate. Each generation, screened up to 600 larvae for RFP, sexed pupae, and quantified egg output to track drive frequency, sex ratio, and population collapse.
• Modeling: Developed deterministic and stochastic discrete-generation models incorporating sex distortion (m), homing rates (d_f, d_m), nonfunctional resistance (R) creation, and potential fitness costs, including an optional damaged X chromosome class from X-shredding. Also implemented a continuous-time population dynamics model with logistic density dependence to compare SDGD vs non-distorting drive impacts on female abundance. Parameterization used experimental fertility, inheritance, and sex-bias estimates.

• Initial SDGDs at fertility loci: Observed super-Mendelian inheritance (mean 79% for SDGD^011377; 98% for SDGD^005958) and male-biased progeny (92–94%) from heterozygous males, but substantial fitness costs (reduced female and, in some cases, male fertility) prevented spread in cages seeded at 12.5% allelic frequency; SDGD^005958 disappeared by generation 2, SDGD^011377 persisted ~8 generations with modest sex-bias (~65% males).
• Optimized SDGDdsx performance:
  • Fertility: Male heterozygotes showed no significant fertility reduction vs wild type (larvae: 126.7 ± 50.7 vs 140.8 ± 40.8; P = 0.39). Female heterozygotes had reduced viable offspring (98.8 ± 63 vs 140.8 ± 40.8; ~37% reduction; P = 0.012) but remained fertile. No significant maternal deposition effect on progeny fertility.
  • Inheritance and sex-ratio bias: Strong super-Mendelian inheritance from heterozygous parents: 96.0% ± 0.08% (males) and 99.9% ± 0.01% (females) RFP+ progeny. Heterozygous SDGDdsx males produced markedly male-biased progeny (mean ~93.1% ± 0.08% males); sex distortion was stably transmitted across generations and independent of parental origin of the construct.
• Cage invasion and suppression: In four independent cage populations (n=600 each), SDGDdsx spread rapidly to 100% allelic frequency between 4 and 12 generations. Populations with 25% initial allelic frequency collapsed by generations 5 and 6; those with 2.5% initial allelic frequency collapsed by generations 9 and 13, with progressive male bias and declining egg output preceding elimination.
• Modeling outcomes: Stochastic simulations (10,000 runs) predicted population collapse in 93% (10% heterozygote release) and 98% (50% release) within 30 generations. Deterministic analyses showed outcomes depend on heterozygote fertility and male-bias rate; sex distortion increases robustness to reduced female fertility and can maintain high load even when female heterozygote fertility is low. No functional resistance at the dsx target site was observed experimentally.
• Comparative advantages (Table 1): SDGDdsx combines rapid spread, strong sex distortion (~93% males), high homing rates (≈96–100%), population suppression from heterozygotes, and absence of selected resistance in cages, outperforming prior autosomal sex distorters and non-distorting drives in robustness and predicted time to impact.

Coupling an X-shredding sex distorter with a homing drive at a functionally constrained dsx site yields an autosomal SDGD that rapidly biases sex ratios toward males while driving itself to high frequency, leading to population collapse without detectable functional resistance. Targeting dsx both curtails the emergence of resistance (due to strong functional constraint and lack of selection for resistant alleles in males) and reduces vector competence by producing biting-incompetent intersex females when homozygous nonfunctional alleles arise. The sex-distortion component synergizes with the drive, enabling spread and imposing high population load even when female heterozygote fertility is reduced, thereby making outcomes less sensitive to uncertain fitness costs. Modeling and cage experiments align, showing faster reduction of transmission-competent females versus non-distorting drives, and suggesting SDGDdsx can achieve robust suppression or elimination under a range of realistic parameters. Component redundancy further enhances robustness: loss of I-PpoI leaves an effective dsx gene drive; loss of drive components still leaves a functional sex distorter producing male-biased progeny.

This study demonstrates an effective autosomal sex-distorter gene drive in Anopheles gambiae by integrating an optimized X-shredding I-PpoI with a CRISPR homing drive targeting a constrained dsx locus. The SDGDdsx achieves strong male bias, high transmission, rapid spread, and consistent population collapse in laboratory cages without selecting functional resistance at the target site. These properties, together with modeling that predicts faster reduction of biting females and robustness to fitness variation, position SDGDdsx as a promising tool for malaria vector control. Future work should evaluate performance and ecological safety under semi-field and field conditions, refine parameter estimates for fitness effects and sex bias in natural settings, monitor for unintended resistance or ecological impacts, and develop deployment strategies and governance frameworks for phased implementation.

• Fitness dependence: Outcomes are sensitive to heterozygote fertility and male-bias rates; deterministic models indicate possible intermediate equilibria rather than fixation when fertility costs are high, which could shift suppression vs elimination outcomes in the field.
• Laboratory conditions: Results derive from contained cage populations with controlled demography; extrapolation to complex field ecologies (migration, seasonality, density dependence, mating structure) is uncertain.
• Prior construct fitness costs: Initial SDGDs at other loci exhibited severe fitness costs (from promoter choice and I-PpoI expression levels), underscoring the importance of construct design and genomic context; although optimized in SDGDdsx, context-specific effects could arise in different genetic backgrounds.
• Modeling assumptions: Models assume no loss of sex-distorter function, no X-shredding resistance, and particular repair rates; deviations in natural populations could alter dynamics.
• Measured resistance scope: Absence of functional resistance at dsx was observed in cages; rare resistance mechanisms might still emerge over longer timescales or larger populations.