Genomic insertion locus and Cas9 expression in the germline affect CRISPR/Cas9-based gene drive performance in the yellow fever mosquito Aedes aegypti

Abstract

ABSTRACTThe yellow fever mosquito Aedes aegypti is a major vector of arthropod-borne viruses, including dengue, chikungunya, and Zika. A novel approach to mitigate arboviral infections is to generate mosquitoes refractory to infection by overexpressing antiviral effector molecules. Such an approach requires a mechanism to spread these antiviral effectors through a population, for example, by using CRISPR/Cas9-based gene drive systems. Here we report an autonomous single-component gene drive system in Ae. aegypti that is designed for persistent population replacement. Critical to the design of a single-locus autonomous gene drive is that the selected genomic locus be amenable to both gene drive and the appropriate expression of the antiviral effector. In our study, we took a reverse engineering approach to target two genomic loci ideal for the expression of antiviral effectors and further investigated the use of three promoters for Cas9 expression (nanos, ß2-tubulin, or zpg) for the gene drive. We found that both promoter selection and genomic target site strongly influenced the efficiency of the drive, resulting in 100% inheritance in some crosses. We also observed the formation of inheritable gene drive blocking indels (GDBI) in the genomic locus with the highest levels of gene drive. Overall, our drive system forms a platform for the further testing of driving antipathogen effector genes through Ae. aegypti populations.Significance statementCRISPR/Cas9 based gene drives for the purposes of population replacement have been developed in Anopheles spp. and self-exhausting split drives have been developed in Ae. aegypti. While these studies show a high level of gene drive, they are exclusively targeted to eye pigmentation genes, which restricts the available number of genomic loci for integration. Given that the genomic locus strongly influences the expression of transgenes, the goal of our work was to migrate an autonomous CRISPR/Cas9 gene drive system into genomic loci known to allow for strong antiviral gene expression in Ae. aegypti, allowing the testing of CRISPR/Cas9 based gene drives to be independent of eye marker genes.