Targeted gene inactivation in Anopeles gambiae via artificial nucleases

描述（由申请人提供）：相对于目前在模式昆虫如果蝇中可用的丰富的分子工具，遗传操纵昆虫疾病载体如疟疾载体蚊子冈比亚按蚊的能力仍然是基本的。最近，人工核酸酶已经允许在以前缺乏有效工具的物种中进行靶向基因组编辑，例如斑马鱼和大鼠。这些嵌合核酸酶联合收割机将可编程的序列特异性DNA结合结构域与非特异性核酸酶结构域组合，以在所需基因组基因座处产生双链断裂，其在不精确地修复时可导致基因失活（“敲除”）。如果这些损伤在胚胎生殖系内产生，则靶向突变等位基因的繁殖是可能的。为了使这种方法为冈比亚按蚊，我们将设计和优化一系列定制的核酸酶，靶向一对充分表征的气味受体（AgOr）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种系基因失活方面的效率，目的是为这种生物体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敲除对成年和幼虫阶段蚊子中的化学感觉反应的影响来证明。除了推进我们对这一重要疾病载体中化学感受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的相关载体物种中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.