Optimization of low-threshold Cas9-based gene drive systems to introduce Zika virus resistance in Aedes aegypti

项目摘要 埃及伊蚊是世界上影响人类健康的节肢动物传播病毒（虫媒病毒）的主要媒介。 热带，如寨卡病毒、基孔肯雅病毒和登革热病毒。寨卡病毒（ZIKV）引起了最近的重大 新世界半球的虫媒病毒爆发作为一种广泛传播的，生活在热带地区的媒介， 区域，Ae.埃及变得越来越难以控制。许多种群已经对 常用的杀虫剂增加了对替代控制策略的需求。一种这样的替代方案是 使用基因改造的抗病毒蚊子来破坏虫媒病毒疾病周期，然而， 这种方法需要一种在群体内传播和固定转基因的机制。新开发基因 驱动（GD）系统能够做到这一点，因为它们以超孟德尔方式偏向基因的遗传。GD 系统可以大致分为人口抑制和人口替代，前者 导致群体崩溃，后者导致转基因在群体中的固定。人口 替代是一个仍在发展中的概念，需要两个遗传成分：一个强大的抗病毒药物， 效应器和强大的GD系统。由于抗病毒效应基因的GD连锁传播，靶向病毒- 感病野生种群将转化为抗病毒种群。最近，我们开发了一种抗病毒药物 ZIKV基因是通过触发蚊子的RNAi途径特异性靶向ZIKV的效应基因。在此，我们寻求 联合收割机将抗ZIKV效应子与基于Cas9的低阈值归巢GD组合，迄今为止， 为Ae开发。埃及人。这些GD依赖于种系中的同源依赖性DNA修复，然而， 在种系外表达Cas9可以导致GD抗性插入缺失。GD阻断插入缺失可以是 通过优化Cas9表达或通过使用能够 当这些插入缺失出现时除去它们。在四个具体目标（SA）中，我们建议为Ae开发。aegypti 与抗ZIKV效应子连接的可靠的低阈值GD系统：1）鉴定用于以下的最佳启动子： 在不同基因组基因座的单组分GD中的Cas9表达; 2）设计和产生新的GD（ClvR， 3）开发用于双GD系统的种系：：Cas9基因融合体，以允许 4）监测优化的GD系统的活性（SA. 1 -3） 通过非重叠小笼研究在多代的野生型样蚊子中进行。四个SA 本申请将确定Ae的GD设计。埃及人，这将允许强大的新特性的引入， 例如对ZIKV的抵抗力，进入野生蚊子种群。

Project Summary Aedes aegypti is the principal vector of arthropod-borne viruses (arboviruses) affecting human health in the tropics, such as Zika, chikungunya, and dengue viruses. Zika virus (ZIKV) caused the most recent major epidemic arbovirus outbreak in the New World Hemisphere. As a wide-spread, peridomestic vector in tropical regions, Ae. aegypti has become ever more difficult to control. Many populations have become resistant to commonly used insecticides increasing the need for alternative control strategies. One such alternative is the use of genetically-modified virus-resistant mosquitoes to disrupt the arboviral disease cycle, however this approach requires a mechanism to spread and fix the transgene within a population. Newly developed gene drive (GD) systems are capable of this, as they bias the inheritance of genes in a super-Mendelian fashion. GD systems can be broadly classified into population suppression and population replacement, with the former resulting in population collapse, and the latter resulting in the fixation of a transgene in a population. Population replacement is a concept still under development, and requires two genetic components: a strong antiviral effector, and a robust GD system. Due to the GD-linked spread of the anti-viral effector gene, a targeted virus- susceptible wild population would be converted into a virus-resistant one. Recently, we developed an anti-viral effector gene that specifically targets ZIKV by triggering the mosquito’s RNAi pathway. Here, we seek to combine the anti-ZIKV effector with low-threshold homing GDs based on Cas9, which so far, have not been developed for Ae. aegypti. These GDs rely on homology dependent DNA repair in the germline, however the expression of Cas9 outside of the germline can lead to GD-resistant indels. GD-blocking indels can be mitigated through the optimization of Cas9 expression, or through the use of novel GD architectures capable of removing such indels when they arise. In four Specific Aims (SA), we propose to develop for Ae. aegypti reliable low-threshold GD systems which are linked to an anti-ZIKV effector: 1) Identify the optimal promoter for Cas9 expression in single-component GD at different genomic loci; 2) Design and generate novel GD (ClvR, HomeR) variants that remove indels; 3) Develop germline::Cas9 gene fusions for a dual-GD systems to allow for the independent drive of the anti-ZIKV effector; 4) Monitor the activity of the optimized GD systems (SA.1-3) in wild-type like mosquitoes via non-overlapping small cage studies over multiple generations. The four SA of this application will identify a GD design for Ae. aegypti that will allow for the robust introduction of new traits, such as resistance to ZIKV, into wild mosquito populations.