Advancements in Genetic Engineering
Open Access

Development of Tissue-Specific Gene Drives for Mosquito Population Control Using Split-Drive Systems

Angela Nossa^{* *}Correspondence: Angela Nossa, Department of Biotechnology and Bioengineering, University of Lille, Lille, France, Email:

Author info »

Description

Vector-borne diseases transmitted by Aedes aegypti mosquitoes, including dengue, zika, and chikungunya, pose significant public health challenges globally. Gene drive technologies offer potential solutions for mosquito population control through the spread of engineered genetic elements that bias inheritance patterns. However, concerns regarding containment and reversibility have limited field applications. This research develops tissue-specific split-drive systems that restrict gene drive activity to reproductive tissues while incorporating multiple safeguard mechanisms for enhanced biosafety.

The split-drive architecture separates Cas9 expression and guide RNA expression into distinct genetic circuits, with Cas9 under control of the germline-specific nanos promoter and gRNAs controlled by the vasa promoter. This design ensures drive activity only occurs in reproductive tissues, preventing somatic expression that could lead to unintended effects. The target gene selected was the doublesex (dsx) gene, which is essential for sex determination and reproductive development in mosquitoes.

Mosquito-borne diseases such as malaria, dengue, zika, and chikungunya continue to pose serious global health challenges, particularly in tropical and subtropical regions. Traditional vector control methods, including insecticides and environmental management, have had limited long-term success due to insecticide resistance and operational constraints. In response, gene drive technologies have emerged as innovative tools for mosquito population suppression or modification by biasing the inheritance of specific genes. Among these, tissue-specific split-drive systems represent a refined and safer approach for developing next-generation vector control strategies.

Unlike full gene drives, which can rapidly spread genetic modifications through entire populations, split-drive systems separate the components of the gene drive—typically the Cas9 nuclease and guide RNA—onto different genetic elements. This modular architecture limits the spread of the gene drive unless both components are inherited together. By further incorporating tissue-specific promoters, researchers can restrict Cas9 activity to selected tissues, such as the germline or reproductive organs, thereby enhancing biosafety and targeting precision. This allows for the disruption of genes critical for mosquito fertility, vector competence, or viability without affecting non-target tissues or unintended organisms.

Tissue-specific split-drive systems offer greater ecological containment, reversibility, and regulatory acceptance compared to traditional gene drives. They enable researchers to fine-tune gene expression, reduce fitness costs, and evaluate drive performance in a controlled and responsible manner. This introduction explores the principles, advantages, and potential applications of tissue-specific gene drives using split-drive systems for sustainable mosquito population control and disease reduction.

Laboratory colonies of Aedes aegypti were established and maintained under controlled conditions with appropriate biosafety measures. Microinjection of fertilized eggs was performed using split-drive constructs cloned into piggyBac transformation vectors. Transgenic mosquitoes were identified through fluorescent marker expression and confirmed through PCR analysis. Inheritance patterns were analyzed across multiple generations to assess drive efficiency and stability.

Experimental results demonstrated successful drive activity with 78% inheritance bias in favor of the drive allele, significantly exceeding the 50% expected under Mendelian inheritance. Importantly, the tissue-specific expression pattern was confirmed through immunofluorescence analysis, with Cas9 protein detected only in germline cells and not in somatic tissues. Phenotypic analysis revealed that homozygous drive mosquitoes exhibited reduced fertility and altered sex ratios, consistent with dsx disruption.

Population modeling studies indicated that the split-drive system could effectively suppress mosquito populations while maintaining spatial containment due to the reduced drive efficiency compared to full-drive systems. Crucially, the reversibility mechanism incorporated into the design allows for drive neutralization through the introduction of resistant alleles that block Cas9 activity.

Conclusion

Tissue-specific split-drive systems offer a promising approach for mosquito population control with enhanced biosafety features. The demonstrated drive efficiency, spatial containment potential, and reversibility mechanisms address key concerns regarding gene drive applications. This work provides a foundation for developing responsible gene drive technologies that balance public health benefits with environmental safety considerations. Environmental risk assessment protocols were established to evaluate potential ecological impacts before any field testing.