Targeted gene inactivation in Anopeles gambiae via artificial nucleases

描述(由申请人提供)：与目前在果蝇等模式昆虫中可用的丰富分子工具相比，对疟疾媒介蚊子冈比亚按蚊等昆虫病媒进行基因操作的能力仍然很低。最近，人工核酸酶允许对以前缺乏有效工具的物种进行有针对性的基因组编辑，如斑马鱼和老鼠。这些嵌合核酸酶将可编程的序列特异性DNA结合域与非特异性核酸酶结构域结合在一起，在所需的基因组位置产生双链断裂，当不精确修复时，可能导致基因失活(“敲除”)。如果这些损伤是在胚胎生殖系内产生的，靶向突变等位基因的传播是可能的。为了使这种方法适用于冈比亚按蚊，我们将设计和优化一系列针对气味受体(AGOR)基因的定制核酸酶，这些基因在嗅觉信号转导中发挥重要作用。在这些研究中，我们将比较两种不同的可编程核酸酶平台在促进按蚊生殖系基因失活方面的效率，目的是为这种生物建立一种强大的反向遗传方法。这一策略的实用性将通过评估不同的agor基因敲除对成虫和幼虫阶段蚊子化学感觉反应的影响而得到证明。除了提高我们对这一重要疾病媒介的化学感觉信号转导的基本知识外，拟议的研究如果成功，将为这些工具和相关基因修饰工具在实验室应用于按蚊和广泛的相关媒介物种提供基础。 公共卫生相关性：尽管进行了数十年的研究，但通过蚊子叮咬传播人类疟疾是全球死亡率和发病率的主要原因。这个项目的重点是开发基因工具，这些工具将大大促进蚊子的研究，并提供对蚊子嗅觉的更详细的了解。这些进展最终可能导致开发新的化学品和方法，以减少疟疾和其他蚊媒疾病的传播。

DESCRIPTION (provided by applicant): The ability to genetically manipulate insect disease vectors such as the malaria vector mosquito Anopheles gambiae remains rudimentary relative to the abundance of molecular tools that are currently available in model insects such as Drosophila. Recently, artificial nucleases have allowed targeted genome editing in species, such as the zebrafish and rat, that previously lacked effective tools. These chimeric nucleases combine a programmable sequence-specific DNA-binding domain with a non-specific nuclease domain to generate a double strand break at a desired genomic locus, which when imprecisely repaired can result in gene inactivation ("knockouts"). If these lesions are generated within the embryonic germline the propagation of targeted mutant alleles is possible. In order to enable this approach for Anopheles gambiae, we will design and optimize a series of custom nucleases targeting a pair of well-characterized odorant receptor (AgOr) genes that play essential roles in olfactory signal transduction. In these studies, we will compare two different programmable nuclease platforms for their efficiency in promoting gene inactivation in the Anopheles germline, with the goal of establishing a robust reverse genetic approach for this organism. The utility of this strategy will be demonstrated by evaluating the effect of various AgOr knockouts on chemosensory responses in adult and larval stage mosquitoes. In addition to advancing our basic knowledge of chemosensory signal transduction in this important disease vector, the proposed studies, if successful, should provide a basis for the laboratory- based application of these and related gene modification tools in Anopheles and a wide range of related vector species. PUBLIC HEALTH RELEVANCE: The transmission of human malaria through mosquito bites is a leading cause of worldwide mortality and morbidity despite decades of research. This project is focused on the developing genetic tools that will significantly enable mosquito research and provide a more detailed understanding of the mosquito's sense of smell. These advances could eventually lead to the development of new chemicals and approaches that reduce the transmission of malaria and other mosquito borne diseases.