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。 热带地区，如寨卡病毒、基孔肯雅热和登革热病毒。寨卡病毒(ZIKV)导致了最近的重大 新大陆半球爆发流行性虫媒病毒。作为一种广泛分布的、在热带地区的周期带病媒 伊利诺伊州埃及伊蚊变得越来越难以控制。许多种群已经对 常用杀虫剂增加了替代防治策略的必要性。一种这样的替代方案是 使用转基因的抗病毒蚊子来扰乱虫媒病毒的疾病周期，然而这 这种方法需要一种在种群中传播和修复转基因的机制。新开发的基因 驱动(GD)系统能够做到这一点，因为它们以一种超级孟德尔式的方式偏向基因遗传。GD 制度大致可分为人口抑制和人口置换，前者 导致种群崩溃，而后者导致种群中的转基因固定。人口 替代是一个仍在开发中的概念，需要两个基因成分：强大的抗病毒药物 效应器，以及强大的GD系统。由于抗病毒效应基因的GD连锁传播，一种靶向病毒-- 敏感的野生种群将转化为抗病毒的种群。最近，我们开发了一种抗病毒药物 通过触发蚊子的RNAi途径来特异性靶向ZIKV的效应基因。在这里，我们寻求 将抗ZIKV效应器与基于Cas9的低门限寻的GDS相结合，到目前为止，还没有 为Ae开发的。埃及伊蚊。这些GDS依赖于生殖系中同源依赖的DNA修复，然而 在种系外表达Cas9可导致抗GD的Indels。Gd-阻止indels可以是 通过优化Cas9表达或通过使用能够 当他们出现的时候，就把他们赶走。在四个具体目标(SA)中，我们建议为Ae开发。埃及伊蚊 与抗ZIKV效应器相连的可靠的低门槛GD系统：1)确定最佳启动子 Cas9在不同基因组位点的单组分GD中的表达；2)设计并产生新的GD(ClvR， 荷马)去除Indels的变体；3)开发生殖系：：Cas9基因融合，以实现双GD系统 用于抗ZIKV效应器的独立驱动；4)监测优化后的GD系统的活性(SA.1-3) 在类似野生型的蚊子中，通过多代非重叠的小笼子研究。的四个SA 此应用程序将标识Ae的GD设计。埃及人将允许强劲地引入新的特征， 例如对ZIKV的抗药性，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.