Nicking endonuclease-mediated gene drive in mosquitoes

 描述（由申请人提供）：登革热流行在过去几十年中有所增加，疾病发病率在过去50年中上升了30倍。这些病毒现在在100多个国家流行，每年发生2亿多例感染，世界人口的50%以上处于危险之中。这种增长的部分原因是人口的巨大增长、人口结构向城市化的转变以及国际贸易和旅行的增加。由于缺乏经批准的登革热特异性预防或治疗药物和疫苗，迫切需要对人和环境安全的新的、具有成本效益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的合理性。长期、可持续的遗传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。用于群体修饰的菌株具有用于此目的的适当性能特征。野生蚊子迁移到一个由经过改造的抗病毒蚊子居住的地区，将通过与当地昆虫交配获得该基因，而进入同一地区的登革热患者将无法感染当地媒介，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种自主的切口核酸内切酶介导的基因驱动（NMG）系统，用于蚊子种系中的位点特异性转基因拷贝数扩增。工作假设是，将有可能工程化Cas9切口核酸内切酶以增加主要登革热载体蚊子埃及伊蚊中每一代的转基因频率。超过孟德尔预期的转基因遗传频率将证明NMG系统的原理。为此，我们的具体目标是：1）合成并验证靶向野生型Ae的适应性NMG系统的位点特异性核酸内切酶活性。埃及犬尿氨酸羟化酶（kh+）基因和2）通过测量NMG转基因的遗传模式分析转基因系以获得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采用控制登革热的人口替代战略产生深远影响。NMG系统的创新设计满足遗传控制技术的许多严格标准，包括仅雄性释放，没有向其他物种的水平转移潜力，以及维持抗病毒效应基因与驱动系统的连接。它还提供了特异性靶向蚊子基因组中任何区域的能力，并通过改变靶向基因序列或利用重组酶介导的插入“对接”位点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.