A Cell-specific modified CRISPR/Cas9 system for conditional gene disruption in Aedes aegypti

PROJECT SUMMARY/ABSTRACT Mosquito-borne disease is a major source of human misery, sickening hundreds of millions and killing hundreds of thousands of people annually. The yellow fever mosquito Aedes aegypti is a primary vector for the viruses that cause dengue, Zika and chikungunya and other diseases. In addition to its medical importance, Ae. aegypti is increasingly used for molecular genetic studies of mosquito vector biology. Understanding Ae. aegypti biology will provide basic science insights into this mosquito vector’s development, physiology and behavior, help inform implementation of control efforts and potentially suggest new control strategies. Improving approaches for genetic investigations in Ae. aegypti will facilitate these efforts. To date, genetic investigations in Ae. aegypti often rely on CRISPR/Cas9-mediated gene targeting to create strains of homozygous mutant animals in which a gene of interest has been disrupted. While extremely valuable, substantial effort is required to create each mutant and the mutants obtained lack cell-specificity. To address these limitations, we propose to establish a cell-specific, modified CRISPR/Cas9 (CmC) toolkit for Ae. aegypti. CmC is designed to decrease the effort required to examine the impact of disrupting a gene of interest, allow the cells in which a gene is acting to be determined and aid the study of essential genes. We propose to achieve these goals in two aims: Aim #1: Introduce the tools for Cell-specific modified CRISPR/Cas9 (CmC) into Ae. aegypti In aim 1a, we design a strategy and build tools for creating mosquito strains that express modified Cas9 transgenes designed to allow optimization of Cas9 levels. In aim 1a2, we use these tools to create mosquitoes that express modified Cas9 transgenes in a specific subset of sensory neurons. In aim 1b, we create transgenes that express multiple gRNAs in a cell-specific fashion to specify the genes targeted. Aim 2: Implement CmC in a subset of sensory neurons as well as pan-neuronally In aim 2a, we test the system, using the tools built to identify an optimal strain which promotes robust cell- specific gene disruption in a subset of sensory neurons, without causing cell death. In aim 2b, we construct mosquito strains that will enable broad neuron-specific gene disruption, which should be of wide utility.