An expanded neurogenetic toolkit to decode olfaction in the African malaria mosquito Anopheles gambiae

Anopheles gambiae uses its sense of smell to hunt humans. We report a two-step method yielding cell-type-specific driver lines for enhanced neuroanatomical and functional studies of its olfactory system. We first integrated a driver-responder-marker (DRM) system cassette consisting of a linked T2A-QF2 driver, QUAS-GFP responder, and a gut-specific transgenesis marker into four chemoreceptor genes (Ir25a, Ir76b, Gr22, and orco) using CRISPR-Cas9-mediated homology-directed repair. The DRM system facilitated rapid selection of in-frame integrations via screening for GFP+ olfactory sensory neurons (OSNs) in G₁ larval progeny, even at genomic loci such as orco where we found the transgenesis marker was not visible. Next, we converted these DRM integrations into T2A-QF2 driver-marker lines by Cre-loxP excision of the GFP responder, making them suitable for binary use in transcuticular calcium imaging. These cell-type-specific driver lines tiling key OSN subsets will support systematic efforts to decode olfaction in this prolific malaria vector.