Project Summary Genome-wide screens using CRISPR-Cas9 technology have revolutionized studies of host-pathogen interactions, leading to identification of many key host cellular factors required for the actions of human pathogens and bacterial toxins. However, this powerful approach has yet to be utilized in insect cells to uncover host factors required for insect-borne pathogens, which are responsible for a long list of infectious diseases such as malaria, Dengue, West Nile, Zika, and Lyme diseases. This is largely due to a technical barrier: the inability to efficiently deliver genome- wide guide RNA library into insect cells. We recently overcame this barrier and developed a genome-wide CRISPR-Cas9 screening method in cultured Drosophila cells. Using this method, and by leveraging the expertise of Dr. Norbert Perrimon’s lab in insect models and of Dr. Min Dong’s lab in bacterial toxins, we carried out extensive preliminary studies, leading to the identification of a potential insect receptor for a member of the major bacterial toxin family known as Tc toxins, demonstrating the power and feasibility of our unbiased genome-wide screen approach. Building on these successes, in Aim 1 we will focus on further development and validation of genome-wide screens with major Tc toxin family members to establish a mechanistic understanding of toxin-receptor interactions and their role in pathogenesis in vitro and in vivo. We further propose to expand our approach to establish the first genome-wide CRISPR-Cas9 screening method and tools in mosquito cells in Aim2, and then utilize this approach to identify key host factors for two novel bacterial toxins that showed selective toxicity on mosquito but not Drosophila cells. The success of our proposal will uncover receptors and key host cellular factors for important bacterial toxins and establish generalizable methods and essential tools for investigating pathogens and toxins at genome-wide scale in insect cells relevant to transmitting human infectious diseases.