Sex-chromosome drives, genetic elements that skew chromosome segregation during meiosis, are over-represented in progeny and cause unbalanced sex ratios in heterogametic species, potentially leading to population suppression or extinction. However, their characterization is rare due to evolutionary conflicts with the genome resulting in autosomal suppressors or resistant sex chromosomes. Mathematical modeling predicts that driving sex distorters, in the absence of resistance, will spread and cause population collapse. This has been observed in laboratory *Drosophila* and in the field with *Drosophila neotestacea*. Therefore, sex-distorter drives could be harnessed for invasive pest or vector control. While Y-chromosome drives are less common, they are attractive for mosquito vector control because they directly reduce disease-transmitting females. Synthetic sex distorters have been created in *A. gambiae* using nucleases like I-Ppol or CRISPR-Cas9, which cleave X-chromosome sequences, favoring Y-chromosome gametes. However, converting these into Y-chromosome drives has proven unsuccessful due to meiotic sex-chromosome inactivation. A previous study showed a gene drive targeting the *dsx* gene reached 100% frequency and crashed a mosquito population without resistance. The researchers hypothesized that an autosomal male-biased sex distorter, coupled with a gene drive, could circumvent meiotic sex-chromosome inactivation, leading to quicker disease transmission reduction and enhanced robustness. This study reports the design and validation of such an SDGD system.

The study builds upon existing research on sex-chromosome drives and gene drives in disease vectors. Previous work demonstrated the potential of using synthetic sex ratio distortion systems, employing site-specific nucleases like I-Ppol or CRISPR-Cas9 to target X-chromosome sequences in *A. gambiae*, leading to a male-biased sex ratio. However, these approaches faced limitations due to meiotic sex chromosome inactivation, preventing the creation of effective Y-chromosome drives. The use of CRISPR-Cas9 gene drives targeting female fertility genes had shown promise, but the study aimed to improve upon these methods by creating an SDGD system that combines the effects of a gene drive and a sex distorter for enhanced efficacy and robustness. The authors cite previous research on the *dsx* gene as a suitable target for gene drives, due to its functional constraints and potential to effectively reduce the vector competence of the population. They also reference studies modelling the potential of gene drives for malaria control and studies exploring the role of various promoters in driving gene expression. The researchers explicitly compared their SDGD approach to earlier gene drives and autosomal sex-distorter systems, highlighting the potential advantages of their approach.

The researchers designed an SDGD system by combining a CRISPR-based gene drive targeting a haplosufficient female fertility gene with the X-chromosome-shredding I-Ppol endonuclease. Mathematical modeling was used to predict the spread of this SDGD. Distinct *A. gambiae* SDGD strains targeting three haplosufficient genes were generated and assessed for drive activity and sex ratio bias in progeny. The initial SDGD constructs showed high male bias but suffered from reduced fertility due to ectopic expression of the vasa promoter. To address this, the vasa promoter was replaced with the zpg promoter, and the beta2-tubulin promoter was modified to reduce I-Ppol expression. A further optimized SDGD construct (SDGD^dsx) targeting the *dsx* gene was created, exhibiting no impact on heterozygote fertility. The SDGD^dsx's performance was assessed in terms of inheritance, sex bias, and fertility in cage experiments. Deterministic and stochastic discrete-generation models were developed to predict SDGD^dsx spread into mosquito populations, simulating releases at different initial frequencies. The models considered various parameters such as the rate of male bias, fertility of heterozygous females and males, and the development of non-functional resistance alleles. These models were then tested against experimental data obtained from cage experiments where SDGD^dsx heterozygotes were released into caged populations of 600 mosquitoes. Larvae were screened for the presence of a fluorescent marker, and sex ratios were determined across generations. Continuous-time population dynamics modeling was also employed to assess the impact of SDGD on population size in the field. The study performed detailed statistical analysis of the experimental data, comparing the performance of SDGD^dsx to previous gene drive approaches.

The SDGD^dsx construct effectively functioned as an autosomal gene drive, rapidly biasing the sex ratio towards males and causing population collapse in cage experiments. No functional mutations blocking the spread of the distorter were observed at the target *dsx* site, supporting the choice of this site as a target for gene-drive solutions. The SDGD^dsx showed no measurable impact on the fertility of heterozygotes. Super-Mendelian inheritance of the construct was observed from both male and female heterozygotes. Mathematical models accurately predicted the rapid spread and population collapse observed in cage experiments. The SDGD^dsx demonstrated superior performance compared to previous gene drives or autosomal sex-distorter systems, particularly in terms of efficacy, robustness, and speed of impact on disease transmission. The study found that the number of transmission-competent (biting) females was reduced much faster by SDGD^dsx than by a standard gene drive targeting the same locus, potentially leading to a significant impact on disease transmission. The researchers noted that although the model predicted complete population elimination in most simulations, there was a possibility of reaching an intermediate equilibrium frequency and population suppression under field conditions, depending on heterozygote fertility. The SDGD system showed robustness, meaning that even if one component (I-Ppol or CRISPR) malfunctioned, the other component could still contribute to population suppression.

The successful implementation of SDGD^dsx in *A. gambiae* demonstrates the potential of this technology for malaria control. The rapid spread and population collapse observed in the cage experiments, coupled with the absence of resistance, suggest that SDGD^dsx could be a highly effective intervention strategy. The study's findings highlight the advantages of SDGD^dsx compared to previous approaches, particularly its robustness, rapid impact on disease transmission, and reduced sensitivity to female fitness costs. The results support the potential for field implementation and the modeling suggests that relatively small numbers of SDGD^dsx mosquitoes could achieve significant population suppression. However, the researchers acknowledge the need for further research and careful consideration of ecological and ethical implications before field trials can be conducted. The study also highlights the importance of comprehensive modeling to predict the outcomes of gene drive interventions and to optimize the design and deployment strategies.

This study successfully demonstrated a novel autosomal male-biased sex-distorter gene drive (SDGD^dsx) in *Anopheles gambiae*. The SDGD^dsx system achieved rapid population suppression in cage experiments without the emergence of resistance, offering a promising strategy for malaria vector control. This approach outperforms previous gene drive methods in terms of efficacy, robustness, and speed of impact on disease transmission. Future research should focus on large-scale modeling and field trials to assess the real-world effectiveness and potential risks of SDGD^dsx, alongside comprehensive ethical considerations and regulatory frameworks.

The study was conducted in a laboratory setting using caged populations of *A. gambiae*. The results may not fully reflect the complexities of real-world environments, which may include factors such as gene flow, diverse genetic backgrounds, and interactions with other species. Extrapolating the findings to field conditions requires further investigation, especially concerning fitness costs of the SDGD in natural populations and potential for unexpected ecological consequences. The models used make several assumptions, such as full fitness in males and heterozygous females, complete sterility in homozygous females, and absence of drive-resistant mutations, which may not fully hold true in natural populations. Although the study found no evidence of resistance in the cage experiments, the long-term potential for resistance development in field settings requires further study. The authors do note that the chosen target locus is expected to minimize resistance, however.