Precision guided SIT for the control of vector-borne disease

项目摘要 数十亿人面临感染病媒传播疾病的风险。仅登革热就造成9000万人感染 与许多病媒传播疾病一样，目前没有药物或疫苗可以治疗或预防 这些感染。因此，病媒控制是用于预防病媒传播疾病的主要工具。在 近年来，新的载体种群抑制技术已经产生（例如RIDL和Wolbachia 基于系统），但这些计划的蚊子生产是劳动密集型的，并且可扩展性有限 和分配。在本研究中，我们将使用功能基因组筛选方法来确定关键性别 确定，女性必需（FE）和男性生育力（MF）基因在登革病毒载体，Ae。埃及人。这些研究 将提高我们对这一重要载体的生物学的理解，它可以用于设计 新的遗传种群抑制方法来控制这种媒介。当这些基因被识别出来， 的特点，我们将把它们纳入精确制导不育昆虫技术（pgSIT）的设计。 技术，试图克服传统的SIT控制策略的局限性。昆虫不育技术 (SIT)是昆虫种群控制的黄金标准，但有许多局限性。我们提出的技术目标是 使用二元CRISPR/Cas9系统在Ae.埃及病 vector.一个系将靶向一个或多个雌性必需FE基因和一个或多个MF基因，而另一个系将靶向一个或多个雌性必需FE基因和一个或多个MF基因。 线将表达Cas9。当这两个品系杂交时，它们产生不育的雄性后代， 进入人口抑制计划。为了生成这些线，最初我们将表征>40 候选FE和MF基因A.在我们先前的研究中， Cas9的表达。这些基因最初将通过转录组学、比较基因组学和基因组学来选择。 基因组学和功能基因组学研究。表现出一致的FE或MF表型的基因靶标将 转基因Ae.表达靶向这些基因的向导RNA（gRNA）的埃及系。这些 然后将品系与多个Cas9品系杂交，并且将每个品系及其Fl后代的适合度进行比较。 经过几代人的努力，以确保人口稳定。设计和整合这些 然后将改变和优化转基因以促进改进的、稳定的和一致的表型。这些 优化实验还将解决关于致死双等位基因嵌合的多个基本问题， 这种现象被认为是推动pgSIT在D.和内源性Cas9表达 系统，包括转基因表达时间和转基因位置对长期稳定性的影响 的线条。然后将在适应度和小群体笼研究中评价最佳设计和基因。 最后，我们的目标是确定新的FE和MF基因，使我们能够更好地了解蚊子的生物学 这使我们能够创建一个遗传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.