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的分子工具相比，基因操纵昆虫疾病媒介（如疟疾媒介冈比亚按蚊）的能力仍然处于初级阶段。最近，人工核酸酶已经允许在斑马鱼和大鼠等物种中进行靶向基因组编辑，这些物种以前缺乏有效的工具。这些嵌合核酸酶将可编程序列特异性dna结合域与非特异性核酸酶结合域结合，在所需的基因组位点上产生双链断裂，当不精确修复时可能导致基因失活（“敲除”）。如果这些病变是在胚胎种系内产生的，则有可能繁殖目标突变等位基因。为了使这种方法适用于冈比亚按蚊，我们将针对一对在嗅觉信号转导中起重要作用的气味受体（AgOr）基因设计和优化一系列定制核酸酶。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种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。该策略的实用性将通过评估各种AgOr敲除对成年和幼虫期蚊子化学感觉反应的影响来证明。除了提高我们对这一重要病媒的化学感觉信号转导的基本知识外，拟议的研究如果成功，将为这些和相关基因修饰工具在按蚊和广泛相关病媒物种的实验室应用提供基础。

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.