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

项目摘要 亚洲虎蚊，白纹伊蚊，是一种攻击性强的叮咬人类的蚊子，是一种合格的媒介 登革热、基孔肯雅和寨卡病毒以及多种北美本土脑炎病毒。的 在过去的30年里，这种蚊子从亚洲本土迅速传播到地球仪， 作为最近登革热和基孔肯雅病毒爆发的主要媒介， 突出的公共卫生问题。本提案的目标是生产和测试一种灵活、高效的 基于CRISPR-Cas9的精确基因组编辑系统在A.白纹伊蚊我们建议生产多个 具有稳定种系表达的化脓性链球菌Cas9蛋白（Cas9）的转基因系， 原生A.白纹伊蚊启动子。我们的理由是内源性表达的Cas9导致更高的编辑率， 更高的技术效率和显着降低的基因组编辑成本相对于注射外源性 Cas9的来源我们先前在另一种密切相关的媒介伊蚊中的工作支持了这一理论 埃及，其中内源性Cas9系的产生不仅改善了这种细胞的功能性遗传能力， 载体，而且还为基于基因的控制技术的发展提供了基础。在目标1中，我们 将产生具有天然A的转基因品系。驱动Cas9表达的白纹伊蚊启动子序列和 荧光报告基因。这些线路将根据最近的“组学”数据在A。白纹伊蚊和 我们在另一个密切相关的载体上的广泛工作。将测试具有不同Cas9启动子和 具有最稳健和稳定的Cas9表达的插入位点和细胞系将在以下研究中进行更广泛的评估： 单基因和多基因敲除研究。在目标2中，我们将设计和测试小的指导RNA，以靶向多个 与A.包括用于质量控制的潜在表型标记的白纹伊蚊控制 和性别分选应用，性别决定基因和飞行特异性基因用于不育昆虫技术， 基因驱动应用以及基因驱动所关注的必需基因。本文中描述的研究 建议将推进A。白纹伊蚊基因组学研究通过：（1）建立一个快速、高效的 功能遗传学和影响广泛表型的基因的反向遗传筛选， 疾病传播和病媒控制，以及（2）完成必要的第一步，开发新的工具， 病媒控制，如遗传性别鉴定和基因驱动。

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.