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

DNA修复途径的选择及其在埃及伊蚊基因组靶向编辑中的意义

• 批准号: 10083695
• 负责人: Zach N. Adelman
• 金额: $ 42.54万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-03 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10083695
• 关键词: Adult; Aedes; Aedes aegypti genome; Alleles; Arboviruses; Behavior; Biological; Biological Assay; Biological Models; Biology; Bombyx; CRISPR/Cas technology; Chemicals; Chikungunya virus; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Culicidae; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA-PKcs; Data; Defect; Dengue; Development; Disease Vectors; Double-Stranded RNA; Embryo; Evolution; Female; Fertility; Funding; G22P1 gene; Generations; Genes; Genetic; Genetic Recombination; Genetic Research; Genome; Heat-Shock Response; Homing; Human; Immunity; Individual; Infrastructure; Interspersed Repetitive Sequences; Knock-in; Knowledge; Ku70 protein; Lesion; Life; Link; Longevity; Measures; Meiosis; Mosquito Control; Organism; Pathway interactions; Pharmaceutical Preparations; Phenotype; Physiology; Plasmids; Play; Population; Population Replacements; Predisposition; Process; Proteins; Public Health; Pupa; Reagent; Repetitive Sequence; Resistance; Role; Sequence Homology; Severities; Sex Differences; Shapes; Site; Source; Structure; Testing; Time; Transgenic Organisms; XRCC5 gene; Yellow Fever; ZIKA; base; design; endonuclease; experimental study; fly; gene drive system; genome editing; improved; insertion/deletion mutation; insight; loss of function; malaria transmission; male; mutant; nuclease; pathogen; preference; prevent; promoter; repair strategy; repaired; resistance gene; sex; vector; vector control; vector mosquito; viral DNA

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.

埃及伊蚊是登革热、寨卡、黄热病和基孔肯雅病毒的主要传播媒介， 这是一个研究其他携带虫媒病毒的蚊子的模型系统。目前的战略 基于抑制蚊子种群的方法可能有效，但价格昂贵， 健全的公共卫生基础设施。将病原体抗性基因渗入的能力 长期以来，人们一直在寻找蚊子种群作为破坏登革热的潜在替代品， 疟疾传播，其中资金和基础设施是蚊子有效传播的限制因素 控制有效的位点特异性核酸酶如归巢酶的研究进展 核酸内切酶和CRISPR/Cas9先进的技术解决方案，以实现这种超 孟德尔渐渗，然而，仍然存在与显性相关的问题， 末端连接过程阻止转基因序列的整合和传播。在这 项目，我们的目标是更好地了解蚊子的DNA修复选择，并制定策略， 在双链DNA断裂诱导后增加基于同源性修复的速率。在 目的1我们将确认各种潜在的末端连接因子在DNA修复中的作用， 成功的女性发展，而在目标2中，我们进行临时控制的救援 实验，以确定最关键的时间在发展过程中的结束连接因素。 最后，在目标3中，我们将评估端部连接因素的损失对各种形式的 同源依赖性修复从这些实验中获得的知识将进一步提供信息， 用于载体控制的基因驱动策略的发展，以及提供对 对蚊子的发育和进化至关重要。