Development of precision genome editing tools in Ae. albopictus for functional genetics and mosquito control technologies

开发 Ae 中的精确基因组编辑工具。

• 批准号: 10362718
• 负责人: Omar Sultan Akbari
• 金额: $ 19.75万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-03 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10362718
• 关键词: Aedes; Affect; American; Arboviruses; Area; Asian; Automobile Driving; CRISPR/Cas technology; Chikungunya virus; Communities; Culicidae; Custom; DNA sequencing; Data; Dengue Virus; Development; Disease Outbreaks; Disease Vectors; Encephalitis Viruses; Essential Genes; Eye Color; Foundations; Future; Genes; Genetic; Genetic Screening; Genetic study; Genome; Genome engineering; Genomics; Goals; Guide RNA; Human; Injections; Knock-out; Molecular; Mosquito Control; Pharmacotherapy; Phenotype; Positioning Attribute; Production; Promoter Regions; Proteins; Public Health; Publishing; Quality Control; Reporter; Reproducibility; Research; Research Personnel; Resources; Role; Site; Sorting - Cell Movement; Source; Streptococcus pyogenes; System; Technology; Testing; Tigers; Tissues; Transgenic Organisms; Vaccines; Virus; Work; Zika Virus; base; cost; design; disease transmission; experimental study; flexibility; functional genomics; gene function; genome editing; genomic tools; genotypic sex; improved; innovation; interest; metropolitan; mutant; novel; novel strategies; phenotypic biomarker; prevent; promoter; protein expression; rational design; reverse genetics; screening; sex; sterile insect technique; tool; transcriptomics; transmission process; vector; vector control

PROJECT SUMMARY The Asian tiger mosquito, Aedes albopictus, is an aggressive human-biting mosquito that is a competent vector of the dengue, chikungunya and Zika viruses as well as multiple native North American encephalitis viruses. The rapid spread of this mosquito from its native Asian range across the globe during the last 30 years and its well- established role as a primary vector of recent outbreaks of both dengue and chikungunya viruses represents an outstanding public health concern. The goal of this proposal is to produce and test a flexible and efficient precision genome editing system based on CRISPR-Cas9 in A. albopictus. We propose to produce multiple transgenic lines with stable germline expression of the Streptococcus pyogenes Cas9 protein (Cas9) driven by native A. albopictus promoters. Our rationale is that endogenously expressed Cas9 leads to higher editing rates, greater technical efficiency and dramatically decreased costs of genome editing relative to injection of exogenous sources of Cas9. This rationale is supported by our previous work in another closely related vector, Aedes aegypti, where the production of endogenous Cas9 lines not only improved functional genetic capabilities in this vector, but also provided the foundation for the development of genetic-based control technologies. In aim 1, we will produce transgenic lines with native A. albopictus promoter sequences driving Cas9 expression and fluorescent reporters. These lines will be rationally designed based on recent “omics” data in A. albopictus and our extensive work in another closely related vector. Lines will be tested that have different Cas9 promoters and insertion sites and lines with the most robust and stable Cas9 expression will be more extensively evaluated in single and multi-gene knockout studies. In aim 2, we will design and test small guide RNAs to target multiple genes of potential relevance to A. albopictus control including potential phenotypic markers for quality control and sex sorting applications, sex determinate genes and flight specific genes for sterile insect techniques and gene drive applications as well as essential genes of interest for gene drives. The research described in this proposal will advance A. albopictus genomics research by: (1) establishing a platform for rapid and efficient functional genetics and reverse-genetic screens of genes affecting a wide range of phenotypes relevant to disease transmission and vector control, and (2) completing a necessary first step to develop novel tools for vector control such as genetic sexing and gene drive.

项目摘要 亚洲老虎蚊子艾德斯白opotus是一种侵略性的人类吸引蚊子 登革热，基孔肯亚和寨卡病毒以及多种北美脑炎病毒。 在过去的30年中 作为最近爆发的粉丝和基孔肯雅病毒爆发的主要媒介，确定了角色 出色的公共卫生问题。该提案的目的是生产和测试灵活而有效的 基于A. alboptus中的CRISPR-CAS9的精确基因组编辑系统。我们建议产生多个 具有稳定种系为链球菌CAS9蛋白（Cas9）的转基因线。 本地白化曲霉启动子。我们的理由是，内源表达的CAS9导致更高的编辑率， 相对于注入外源性的，基因组编辑的更高技术效率和大幅提高了基因组编辑的成本 CAS9的来源。我们以前在另一个密切相关的向量艾德斯（Aedes）的工作支持了这一理由 埃及，其中内源性CAS9线的产生不仅提高了功能遗传能力 向量，但也为基于遗传的控制技术的发展奠定了基础。在AIM 1中，我们 将产生具有天然白化曲霉启动子序列的转基因线，驱动Cas9表达和 荧光记者。这些线条将根据A. alboptus和 我们在另一个密切相关的矢量中进行的广泛工作。将测试具有不同CAS9启动子和 将在以最强和稳定的Cas9表达表达的插入位点和线路中进行更广泛的评估 单一和多基因敲除研究。在AIM 2中，我们将设计和测试小指南RNA以靶向多个 与白局的潜在相关性基因，包括质量控制的潜在表型标记 以及性排序应用，性别确定的基因和无菌绝缘技术的飞行特定基因 基因驱动应用以及基因驱动的基本基因。在此描述的研究 提案将推进白化曲霉基因组学研究，作者：（1）建立一个快速有效的平台 影响与广泛表型相关的广泛表型的功能遗传学和反遗传筛选 疾病传播和矢量控制，以及（2）完成开发新颖工具的必要第一步 载体控制，例如遗传性别和基因驱动。