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

项目摘要/摘要 蚊媒疾病是人类苦难的主要来源，使数亿人患病并导致死亡 每年有数十万人。黄热病蚊子埃及伊蚊是传播黄热病的主要媒介 导致登革热、寨卡病毒和基孔肯雅热等疾病的病毒。除了它在医学上的重要性，Ae. 埃及伊蚊越来越多地被用于蚊媒生物学的分子遗传学研究。了解Ae. 埃及伊蚊生物学将为这种蚊子媒介的发育、生理和 行为，有助于为控制努力的实施提供信息，并潜在地建议新的控制策略。 改进Ae基因研究的方法。埃及人将促进这些努力。 到目前为止，Ae.埃及伊蚊经常依靠CRISPR/Cas9介导的基因打靶来 创造纯合子突变动物的品系，其中一个感兴趣的基因已经被扰乱。虽然极端 创造每一个突变体都需要付出宝贵的努力，而获得的突变体缺乏细胞特异性。至 为了解决这些限制，我们建议为Ae建立一个细胞特异性的、经修改的CRISPR/Cas9(CMC)工具包。 埃及伊蚊。CMC旨在减少检查干扰目标基因的影响所需的工作量， 允许确定基因在其中起作用的细胞，并帮助研究必要的基因。 我们建议通过两个目标实现这些目标： 目的#1：将细胞特异性改良CRISPR/CAS9(CMC)工具引入Ae。埃及伊蚊 在目标1a中，我们设计了一种策略并构建了用于创建表达经修饰的Cas9的蚊子菌株的工具 转基因设计，以允许优化Cas9水平。在Aim 1a2中，我们使用这些工具来制造蚊子 在特定的感觉神经元亚群中表达修饰的Cas9转基因。在目标1b中，我们创造了转基因 以细胞特有的方式表达多个gRNA，以指定靶向的基因。 目标2：在部分感觉神经元和泛神经元中实施CMC 在目标2a中，我们使用构建的工具来测试系统，以确定促进强大细胞的最佳菌株- 在感觉神经元的一个子集中的特定基因破坏，而不会导致细胞死亡。在目标2b中，我们构建 蚊子菌株将实现广泛的神经元特异性基因破坏，这应该会有广泛的用途。

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.