Nicking endonuclease-mediated gene drive in mosquitoes

 描述(申请人提供)：登革热疫情在过去几十年中有所增加，疾病发病率在过去50年中增加了30倍。这种病毒现在在100多个国家流行，每年有超过2亿人感染，世界上50%的人口处于危险之中。这一增长在一定程度上是由巨大的人口增长推动的，人口结构朝着城市化方向发展，以及国际贸易和旅游的增加。由于缺乏经批准的针对登革热的预防或治疗药物和疫苗，迫切需要对人和环境安全的新的、成本效益高和有效的疾病控制工具。这种需要证明了努力开发控制病毒传播的遗传方法是合理的。长期、可持续的基因控制将需要部署旨在抵御易感蚊子和登革热感染者移民的战略。用于种群改造的菌株具有适合这一目的的性能特征。野生蚊子迁徙到一个由转基因抗病毒蚊子组成的地区，将通过与当地昆虫交配获得这种基因，而迁入同一地区的登革热患者将无法感染当地的媒介生物，因此不会成为其他人的感染源。我们将利用CRISPR/Cas9生物学的分子机制来开发一个自主的缺口内切酶介导的基因驱动(NMG)系统，用于蚊子生殖系中位置特异的转基因拷贝数扩增。工作假设是，有可能设计一种Cas9缺口内切酶，以提高主要登革热媒介蚊子埃及伊蚊每代的转基因频率。超过孟德尔预期的转基因遗传频率将证明NMG系统的原则。为此，我们的具体目标是：1)合成并验证针对野生型Ae的适应性NMG系统的位点特异性内切酶活性。Aegypti Kynurenine羟基酶(kh+)基因和2)通过测量NMG转基因的遗传模式来分析转基因系以寻找自主基因驱动特性的证据。NMG系统的成功开发将对进一步测试和可能最终通过人口替换战略以控制登革热产生深远影响。NMG系统的创新设计符合一些严格的遗传控制技术标准，包括仅释放男性，不向其他物种水平转移潜力，以及保持抗病毒效应基因与驱动系统的链接。它还提供了专门针对蚊子基因组中任何区域的能力，并通过改变目标基因序列或利用重组酶介导的‘对接’位置插入来引入额外的效应基因。

 DESCRIPTION (provided by applicant): Dengue fever epidemics have increased over the last decades and disease incidence has risen 30-fold in the last 50 years. The viruses are now endemic in more than 100 countries and over 200 million infections occur annually with >50% of the world's population at risk. This rise is fueled in part by tremendous population growth, a shif in demographics towards urbanization and increased international trade and travel. The lack of approved dengue-specific prophylactic or therapeutic drugs and vaccines makes urgent the need for new, cost- effective and efficacious disease-control tools that are safe for people and the environment. This need justifies efforts to develop genetic approaches for controlling virus transmission. Long-term, sustainable genetic control will require the deployment of strategies designed to be resilient to the immigration of susceptible mosquitoes and dengue-infected people. Strains for population modification have the appropriate performance features for this purpose. Wild mosquitoes immigrating into a region populated by engineered, virus-resistant mosquitoes will acquire the gene by mating with the local insects, and persons with dengue moving into the same region will not be able to infect the resident vectors, and therefore are not a source for infection of other people. We will exploit the molecular mechanisms of the CRISPR/Cas9 biology to develop an autonomous nicking endonuclease-mediated gene drive (NMG) system for site-specific, transgene copy number amplification in the mosquito germline. The working hypothesis is that it will be possible to engineer a Cas9 nicking endonuclease to increase transgene frequency at each generation in the major dengue-vector mosquito Aedes aegypti. Transgene inheritance frequencies that exceed Mendelian expectations will demonstrate the proof-of-principle for the NMG system. Towards these ends, our Specific Aims are: 1) synthesize and validate site-specific endonuclease activity of an adaptable NMG system targeting the wild-type Ae. aegypti kynurenine hydroxylase (kh+) gene and 2) analyze transgenic lines for evidence of autonomous gene-drive properties by measuring inheritance patterns of NMG transgenes. The successful development of the NMG system will have a profound impact on the further testing and possible final adoption of a population-replacement strategy for controlling dengue. The innovative design of the NMG system meets a number of stringent criteria for genetic- control technologies including male-only releases, no horizontal transfer potential to other species and maintenance of linkage of anti-viral effector genes with the drive system. It also provides the ability to target specifically any region in the mosquito genome and introduce additional effector genes by changing the targeted gene sequence or exploiting recombinase-mediated insertion at a 'docking' site.