CRISPR/Cas9-mediated gene deletion of the ompA gene in an Enterobacter gut symbiont impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes

Symbiotic bacteria are pervasive in mosquitoes and their presence can influence development, reproduction, and immunity of their host. It is evident that environmental and host genetic factors contribute in shaping the microbiome of mosquitoes, but we have a poor understanding regarding how bacterial genetics affects colonization of the mosquito gut. While CRISPR/Cas9 gene editing is a powerful tool to modify bacterial genomes this approach has yet to be applied to insect symbionts. To demonstrate that gene editing can be completed in non-model bacterial species isolated from insects and to investigate the role of bacterial genes in gut colonization, we mutated the outer membrane protein A (ompA) gene of an Enterobacter symbiont using the CRISPR/Cas9 system. The ∆ompA mutant had an impaired ability to form biofilms and poorly infected Ae. aegypti when reared in a mono-association under gnotobiotic conditions. In adults, the mutant had a significantly reduced infection prevalence compared to the wild type or complement strains, while no differences in prevalence were seen in larvae, suggesting bacterial genetic factors are important for adult gut colonization. Integration of genes (antibiotic resistance and fluorescent markers) into the symbiont genome demonstrated this technology can be exploited to develop novel symbiotic control strategies to interfere with arboviral pathogens such Chikungunya, Zika and Yellow fever viruses transmitted by Aedes mosquitoes. Our results shed insights onto the role of ompA gene in host-microbe interactions in Ae. aegypti and confirm that CRISPR/Cas9 gene editing can be employed for genetic manipulation of non-model gut microbes.