Development of a pan-neuronal genetic driver in Aedes aegypti mosquitoes

The mosquito Aedes aegypti is the primary worldwide vector of arboviruses that infect humans, including dengue, Zika, chikungunya, and yellow fever. Recent advances in transgenic technology have yielded important new insight into the biology of this disease vector. The early development of neurogenetic tools, in particular, is beginning to shed light on the neural basis of behaviors that allow Ae. aegypti to thrive in human environments and find and bite human hosts. Despite these advances, a pan-neuronal expression driver remains elusive. Pan-neuronal drivers give researchers genetic access to all neurons and thus provide a critical entry point for circuit dissection. Here, we describe our efforts to generate pan-neuronal drivers in Ae. aegypti via targeted knock-in of in-frame reporter constructs to the native coding sequence of broadly expressed neural genes with CRISPR/Cas9. Two of five attempts were successful, resulting in a Syt1:GCaMP6s strain that expresses synaptically-localized GCaMP in all neurons and a brp-T2A-QF2w driver strain that can be used to drive and amplify expression of any effector in all neurons via the Q binary system. We show that both manipulations broadly and uniformly label the nervous system and have only mild effects on behavior. We envision that these strains will facilitate neurobiological research in Ae. aegypti mosquitoes and provide documentation of both successful and failed manipulations as a roadmap for similar tool development in other non-model species.