Precision guided SIT for the control of vector-borne disease

项目总结 数十亿人面临感染媒介传播疾病的风险。仅登革热一项就导致9000万人感染 每年在全球范围内，就像许多媒介传播的疾病一样，目前没有治疗或预防的药物或疫苗 这些感染。因此，病媒控制是预防病媒传播疾病的主要工具。在……里面 近年来，已经创造了新的媒介种群抑制技术(例如RIDL和Wolbachia 基于系统)，但为这些项目生产蚊子是劳动密集型的，并且可伸缩性有限 和分配。在这项研究中，我们将使用功能性基因组筛选方法来鉴定关键性别 登革热载体中的确定、女性必需(FE)和男性生育(MF)基因。埃及伊蚊。这些研究 将提高我们对这一重要载体生物学的理解，它可以用来指导设计 控制这种媒介的新的遗传种群抑制方法。在鉴定了这些基因之后， 我们将把它们纳入精确引导昆虫不育技术(PgSIT)的设计中。 技术，试图克服传统SIT控制策略的局限性。昆虫不育技术 (SIT)是控制昆虫数量的黄金标准，但有许多局限性。我们建议的技术目标是 在Ae中使用二进制CRISPR/Cas9系统同时敲除FE和MF基因。埃及伊蚊 向量。一条线将针对一个或多个女性必需FE基因和一个或多个MF基因，而另一条线将针对另一条线 LINE将表示Cas9。当这两个品系杂交时，它们就会产生不育的雄性后代，为 释放到人口抑制计划中。要生成这些行，首先我们将描述&gt；40 在我们以前的单一和组合sgRNA筛选试验中，候选的FE和MF基因埃及伊蚊 表征了Cas9的表达。这些基因将通过转录组学进行初步选择，比较 基因组学和功能基因组学研究。表现出一致的FE或MF表型的基因靶点将 被改造成转基因Ae。埃及油菜系表达针对这些基因的引导RNA(GRNA)。这些 然后将品系杂交到多个Cas9品系，每个品系及其F1后代的适合度将是 经过几代人的努力，以确保人口稳定。这些组件的设计和集成 然后，转基因将被多样化和优化，以促进改进、稳定和一致的表型。这些 优化实验还将解决关于致命双等位嵌合体的多个基本问题，即 在黑腹果蝇中发现的推动pgSIT成功的现象和内源Cas9的表达 系统，包括转基因表达时机和转基因位置对长期稳定性的影响 在台词中。然后，将在健康和小种群笼子研究中对最佳设计和基因进行评估。 最后，我们的目标是识别新的FE和MF基因，使我们能够更好地了解蚊子的生物学 这使我们能够创造一种基因SIT系统，改进传统的SIT技术。

PROJECT SUMMARY Billions of people are at risk of contracting vector-borne diseases. Dengue alone causes 90 million infections per year globally and like many vector-borne diseases, currently there are no drugs or vaccines to treat or prevent these infections. Therefore, vector control is the primary tool used for vector-borne disease prevention. In recent years, novel vector population suppression technologies have been created (e.g. RIDL and Wolbachia based systems), but production of mosquitoes for these programs is labor intensive and is limited in scalability and distribution. In this study, we will use a functional genomic screening approach to identify key sex determinate, female essential (FE) and male fertility (MF) genes in the dengue vector, Ae. aegypti. These studies will improve our understanding of the biology of this important vector and it can be used to inform the design of new genetic population suppression methods to control this vector. After these genes are identified and characterized, we will incorporate them into the design of precision guided sterile insect technique (pgSIT) technologies in an attempt to overcome limitations in traditional SIT control strategies. Sterile insect technique (SIT) is the gold standard for insect population control but has many limitations. Our proposed technology aims to simultaneously knock-out FE and MF genes using a binary CRISPR/Cas9 system in the Ae. aegypti disease vector. One line will target one or more female essential FE genes and one or more MF genes and the other line will express a Cas9. When these two lines are crossed, they create sterile, male progeny that are ready for release into a population suppression program. To generate these lines, initially we will characterize >40 candidate FE and MF genes A. aegypti in single and combinatorial sgRNA screening assays in our previously characterized Cas9 expression. These genes will be initially selected through transcriptomics, comparative genomics and functional genomic studies. Gene targets that exhibit consistent FE or MF phenotypes will then be engineered into transgenic Ae. aegypti line expressing guide RNAs (gRNA) targeting these genes. These lines will then be crossed to multiple Cas9 lines and the fitness of each line and their F1 progeny will be determined over many generations to ensure population stability. The design and integration of these transgenes will then be varied and optimized to facilitate improved, stable and consistent phenotypes. These optimization experiments will also address multiple fundamental questions about lethal biallelic mosaicism, a phenomenon identified as driving pgSIT success in D. melanogaster, and endogenous Cas9 expression systems, including the impact of transgene expression timing and transgene location on the long-term stability of the lines. The optimal design and genes will then be evaluated in fitness and small population cage studies. In the end, we aim to identify novel FE and MF genes that will allow us to better understand mosquito biology and which allow us to create a genetic SIT system that improves upon traditional SIT technologies.