Resource of Functional Genomics Screening Platforms

Project Summary Mosquitos are vectors for a wide range of pathogens that kill hundreds of thousands of people each year and sicken many more. Anopheles mosquitos are vectors for malaria, for example, and Aedes and Culex mosquitos can be vectors for more than 30 viral pathogens including Chikungunya virus, Dengue virus, West Nile virus, and yellow fever virus. Genome-wide screening in mosquito cultured cells has strong potential to provide an efficient and effective forward-genetic platforms for studying mosquito biology, including topics such as viral pathogen entry, other host cell-viral pathogen interactions, immune signaling, hormone signaling, insecticide resistance, and resistance to environmental toxicants. In this work, we will establish CRISPR pooled-format screening in five cell lines from four disease vector mosquito species, an Anopheles group mosquito, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus. Specifically, we will develop engineered cell lines, relevant plasmids, genome-wide CRISPR guide RNA libraries, and related resources that facilitate knockout and over-expression screens in cells from these species. For Aedes albopictus, which has more paralogs than other species, we will additionally develop a multiplexed approach to support concurrent knockout of paralogous immune-related genes. The resulting screening resources will make it possible for the first time to perform genome-wide knockout and over-expression screens directly in cell lines from four mosquito species, opening the doors to a broad range of studies by the vector community with potential to efficiently annotate gene function and uncover new biology that can lead to new interventions that limit the spread of disease.