Precision guided SIT for the control of vector-borne disease

精准引导昆虫不育技术用于控制媒介传播疾病

• 批准号: 10087886
• 负责人: Omar Sultan Akbari
• 金额: $ 46.12万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-23 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10087886
• 关键词: Address; Aedes; Alleles; Automobile Driving; Biological Assay; Biology; CRISPR/Cas technology; Candidate Disease Gene; Contracts; Culicidae; Data; Dengue; Dengue Fever; Development; Disease Vectors; Drosophila melanogaster; Embryo; Engineering; Ensure; Entomology; Environment; Essential Genes; Evaluation; Exhibits; Female; Fertility; Fertility Study; Frequencies; Gene Targeting; Generations; Genes; Genetic; Genetic Vectors; Genome; Gold; Guide RNA; Homing; Infection; Infection prevention; Insecta; Knock-out; Lead; Legal; Life; Location; Malaria; Male Sterility; Masculine; Mechanics; Methods; Mosaicism; Mosquito Control; Organism; Pharmaceutical Preparations; Phenotype; Population; Population Control; Population Genetics; Production; Reproducibility; Risk; Series; Site; Sterility; System; Technology; Transgenes; Transgenic Organisms; Vaccines; Vector-transmitted infectious disease; Wolbachia; Work; Yellow Fever; ZIKA; base; combinatorial; comparative genomics; cost; cost effective; design; disorder prevention; egg; endonuclease; experimental study; fitness; functional genomics; improved; innovation; insight; loss of function; male; male fertility; mathematical model; new technology; novel; offspring; prevent; programs; screening; sex; sex determination; sexual dimorphism; sterile insect technique; success; technology/technique; tool; transcriptomics; transgene expression; vector; vector control; vector mosquito; zika fever

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.

项目摘要 数十亿人有患媒介传播疾病的风险。仅登革热就会引起9000万感染 每年在全球范围内和许多媒介传播疾病，目前没有药物或疫苗可以治疗或预防 这些感染。因此，载体控制是用于预防媒介疾病的主要工具。在 近年来，新颖的媒介种群抑制技术已经创建了（例如Ridl和Wolbachia 基于系统），但是为这些程序生产蚊子是劳动密集型的，并且可伸缩性有限 和分布。在这项研究中，我们将使用功能性基因组筛选方法来识别关键性别 登革热载体中的确定，女性必需（Fe）和男性生育能力（MF）基因。埃及。这些研究 将提高我们对这个重要向量的生物学的理解，可以用来告知设计 控制该载体的新遗传种群抑制方法。在确定这些基因之后， 特征是，我们将它们纳入精确引导的无菌昆虫技术（PGSIT）的设计中 技术试图克服传统的SIT控制策略的局限性。无菌昆虫技术 （SIT）是昆虫种群控制的黄金标准，但有许多局限性。我们提出的技术目的 使用AE中的二元CRISPR/CAS9系统同时敲除Fe和MF基因。埃及疾病 向量。一条线将针对一个或多个女性基本Fe基因和一个或多个MF基因，而另一个 线将表示CAS9。当这两条线越过时，它们会创造出无菌的男性后代 释放到人群抑制计划中。要生成这些行，最初我们将表征> 40 候选Fe和MF基因A. A. aegypti在我们以前的单一和组合SGRNA筛选中 表征CAS9表达。这些基因最初将通过转录组学，比较选择 基因组学和功能基因组研究。那么表现出一致的Fe或MF表型的基因靶标将 被设计成转基因AE。 aegypti线表达指导RNA（GRNA）针对这些基因。这些 然后，线将越过多个CAS9线，每条线的适应性及其F1后代将是 确定多代人以确保人口稳定。这些的设计和整合 然后，转基因将被变化和优化，以促进改进，稳定和一致的表型。这些 优化实验还将解决有关致命性双质镶嵌的多个基本问题， 现象被确定为在D. melanogaster中驱动PGSIT成功，并且内源性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 线。然后，最佳设计和基因将在适应性和较小的人口笼研究中进行评估。 最后，我们旨在确定新颖的FE和MF基因，从而使我们能够更好地了解蚊子生物学 这使我们能够创建一个遗传SIT系统，以改进传统的SIT技术。