Characterizing and engineering a dengue refractory phenotype in Aedes aegypti

Dengue viruses infect ~400 million people annually and are transmitted principally by Aedes aegypti. Severe dengue (dengue hemorrhagic fever and dengue shock syndrome) can be fatal, and there are no efficient drugs or vaccines to prevent the disease. Not all Ae. aegypti transmit dengue viruses; in Cali, Colombia, approximately 30% of feral populations are naturally refractory to all four viral serotypes through midgut mechanisms (Cali-MIB), while the remaining 70% are susceptible (Cali-S) and transmit the viruses. We used a combination of molecular biology and bioinformatic methods to identify differences between the refractory and susceptible strains. RNA sequencing, 16S rRNA bacterial profiling, and a genome wide association study (GWAS) were used to identify a subset of genes thought to contribute to the Cali-MIB and Cali-S phenotypes. Genes from this subset that were able to 'flip' the phenotype from susceptible to refractory through RNAi based knockdowns were further tested with gene-editing technology to knock-out these genes using clustered regularly interspaced palindromic repeats (CRISPR) – CRISPR-associated protein 9 (Cas9) guide RNA complexes. This research identified multiple genes we believe contribute to vector competence, created a DNA based assay for identifying Cali-MIB and Cali-S mosquitoes, and edited the germ-line of Ae. aegypti. This information could allow us to create lines of permanently refractory mosquitoes to dampen dengue transmission.