DNA repair pathway choice and significance in targeted genome editing of Aedes aegypti

埃及伊蚊是登革热、寨卡病毒、黄热病和基孔肯雅病毒的主要传播媒介， 用于研究传播虫媒病毒的其他蚊子的模型系统。目前的策略 基于抑制蚊子数量的方法可能有效，但价格昂贵且需要 健全的公共卫生基础设施。将病原体抗性基因渗入到 长期以来，人们一直在寻找消灭蚊子种群作为破坏登革热或 资金和基础设施是疟疾传播有效蚊虫传播的限制因素 控制。最近开发了有效的位点特异性核酸酶，例如归巢酶 核酸内切酶和CRISPR/Cas9先进的技术解决方案来实现这种超级 然而，孟德尔基因渗入仍然存在与优势相关的问题 末端连接过程阻止转基因序列的整合和传播。在这个 项目中，我们的目标是更好地了解蚊子的 DNA 修复选择并制定策略 提高双链 DNA 断裂诱导后基于同源性的修复率。在 目标1 我们将确认各种潜在末端连接因子在DNA修复和 成功的女性发育，而在目标 2 中我们进行时间控制的救援 进行实验以确定末端连接因素开发过程中最关键的时间。 最后，在目标 3 中，我们将评估末端连接因素丢失对各种形式的影响 同源依赖性修复。从这些实验中获得的知识将进一步告知 开发用于病媒控制的基因驱动策略并提供见解 对蚊子发育和进化至关重要的过程。

Aedes aegypti is the main vector of dengue, Zika, yellow fever and chikungunya viruses, and is a model system for studies of other mosquitoes that vector arboviruses. Current strategies based on suppressing mosquito populations can be effective, but are expensive and require a robust public health infrastructure. The ability to introgress pathogen-resistance genes into mosquito populations has long been sought as a potential alternative for disrupting dengue or malaria transmission where funds and infrastructure are the limiting factors in effective mosquito control. The recent development of effective site-specific nucleases such as homing endonucleases and CRISPR/Cas9 advanced technical solutions to achieving such super- Mendelian introgression, however there are still problems associated with the dominance of end-joining processes preventing the integration and spread of transgenic sequences. In this project, we aim to better understand DNA repair choice in mosquitoes and develop strategies to increase rates of homology-based repair following double-stranded DNA break induction. In Aim1 we will confirm the role of various potential end-joining factors in DNA repair and successful female development, while in Aim 2 we perform temporally-controlled rescue experiments to determine the most critical times during development for end-joining factors. Finally in Aim 3 we will assess the impact of loss of end-joining factors on various forms of homology-dependent repair. The knowledge gained from these experiments will further inform the development of gene drive strategies for vector control as well as provide insight into processes critical for mosquito development and evolution.