Homing-based gene drives, using CRISPR-Cas9 technology, offer a promising method for controlling harmful insect populations. These drives aim to reduce pest numbers by targeting genes crucial for fitness, exceeding Mendelian inheritance rates. Focusing on female reproduction or sex ratio manipulation are particularly effective strategies. While gene drives have shown success in laboratory settings with mosquito species, their application to agricultural pests remains largely unexplored. Tephritid fruit flies, such as the medfly (*Ceratitis capitata*), are globally significant agricultural pests, impacting food production. Current control methods, such as the Sterile Insect Technique (SIT), are effective but can be resource-intensive. The medfly is particularly amenable to gene drive development due to existing research on its genetics and mass-rearing techniques, along with recent advances in CRISPR-Cas9 genome editing within the species. Moreover, the medfly exhibits a unique malleability in its sex determination pathway, allowing for the generation of fertile XX males, offering potential for novel sex conversion gene drive strategies. This study aims to establish the feasibility of gene drives in medflies, initially targeting a phenotypic marker gene (*white-eye*), and subsequently focusing on the *transformer* gene to achieve genetic sex conversion. The researchers hypothesized that a gene drive targeting the *transformer* gene, combined with a female sterility gene, would lead to highly effective population suppression.

The researchers review the existing literature on homing gene drives and their application in various insect species, including medically relevant mosquitoes and other agricultural pests. They highlight the success of gene drives in reducing mosquito populations in laboratory settings and the ongoing efforts to establish frameworks for field testing and deployment. They also discuss the history and success of the Sterile Insect Technique (SIT) for tephritid control, emphasizing the existing infrastructure and knowledge base that can facilitate the development of gene drives in these species. The researchers also refer to previous research that established the basic CRISPR/Cas9 tools in *C. capitata*, mapping the sex-determination pathway and showing the possibility of generating XX fertile males. The review sets the stage for exploring the potential of gene drives in medflies, particularly in leveraging the species' unique sex determination system for sex conversion strategies.

The study used CRISPR-Cas9-based gene drive constructs in *C. capitata*. They first evaluated homing efficiency in the *white-eye* gene using three different germline-specific promoter elements (*nanos*, *vasa*, and *zpg*). Transgenic flies were crossed with *white-eye* mutant and wild-type flies, and progeny were analyzed for *DsRed* marker inheritance (indicating gene drive success) and eye pigmentation (indicating Cas9 activity). They then developed a gene drive targeting the *transformer* (*Cctra*) gene to induce sex conversion. Transgenic males were crossed with wild-type females, and progeny were analyzed for *DsRed* expression, sex, and *Cctra* gene sequencing to assess sex conversion efficiency. To further explore sex conversion, they created a dual-gRNA construct targeting both *white-eye* and *Cctra*, aiming to bias inheritance and disrupt *Cctra* in the female germline. Progeny were analyzed for gene drive transmission and sex ratios. Fecundity and fertility assays were performed to evaluate the impact of the gene drives on reproduction. Finally, an agent-based model simulated the sterilising sex conversion (SSC) strategy (co-targeting female fertility and *transformer*) to assess its effectiveness and resistance tolerance compared to canonical gene drive strategies.

The researchers found that *C. capitata* is highly amenable to homing-based gene drives. The *vasa* promoter showed the highest gene drive efficiency in both male and female germ lines in the *white-eye* targeting experiments, achieving super-Mendelian inheritance above 70% in most crosses involving transgenic females. Analysis of eye color phenotypes in the offspring correlated well with Cas9 activity and maternal deposition of Cas9. The gene drive targeting the *Cctra* gene showed high homing rates in males and unexpectedly led to male-biased progeny due to somatic Cas9 activity and sex conversion of females. Although most XX individuals carrying the *tra* drive were infertile intersexes, a fraction were converted into fertile XX males. The dual-gRNA construct targeting both *white-eye* and *Cctra* showed efficient gene drive transmission and a strong male bias in the progeny of transgenic mothers, suggesting a potent maternal effect. Importantly, there was no significant difference in fertility between transgenic and wild-type flies. Modeling of a sterilising sex conversion (SSC) strategy indicated enhanced resistance tolerance compared to traditional gene drive approaches, due to its synergistic targeting of females via sex conversion and female sterility, delaying counterselection.

The study's findings demonstrate the feasibility of using gene drives to control medfly populations. The high efficiency of homing observed, particularly with the *vasa* promoter, suggests that this approach is viable and can be optimized further with improved regulatory elements. The successful generation of a gene drive targeting *Cctra* demonstrates the potential of sex conversion as a population control strategy. The unexpected male bias observed in the progeny of *tra* drive males highlights the importance of considering somatic Cas9 activity. The dual-gRNA construct, and particularly the modeling results, suggest a potentially robust and highly effective strategy for population suppression even in the presence of resistance alleles by co-targeting female fertility and *transformer* function. The lack of negative effects on fecundity and fertility further supports the environmental friendliness of this approach. Future research could focus on optimizing the SSC strategy for increased effectiveness and exploring potential field applications.

This research successfully established homing gene drives in the medfly, showcasing the potential of this technology for agricultural pest control. The demonstration of sex conversion via targeting the *transformer* gene offers a novel strategy. The modeling strongly supports the sterilising sex conversion (SSC) strategy as a highly effective, robust method for population suppression, even in the face of resistance. This approach offers a promising, environmentally friendly and economically viable alternative to traditional pest management strategies. Future work should involve optimizing the gene drives for field deployment, conducting risk assessments, and addressing any ethical considerations.

The study primarily focused on laboratory experiments. While the modeling provided insights into the potential of the SSC strategy, further research is needed to validate these findings in field settings. The somatic activity of Cas9, while contributing to sex conversion, also led to the generation of infertile intersex individuals, potentially affecting the overall efficiency of the *tra* drive. The study did not directly address the long-term evolutionary dynamics of the gene drive system in a natural environment.