Efficient site-specific genetic engineering of the Anopheles gambiae genome

描述(由申请人提供)：冈比亚按蚊基础和应用分子研究面临的最大障碍之一是该物种的转基因效率低得令人望而却步。自20年来首次报道了稳定的种系转化以来，仅有少数转基因植株获得了成功。冈比亚亚纲的线条已经出版。冈比亚按蚊缺乏有针对性的基因敲除和基因替换工具，目前被认为是一项关键的研究需要。在其他系统中，锌指核酸酶(ZFN)是通过诱导双链断裂(DSB)进行靶向基因破坏的非常有效的工具。当不提供同源供体模板时，ZFN诱导的DSB通过非同源末端连接(NHEJ)介导的框架移位导致高效的基因破坏。当DSB发生时，如果有同源模板(如质粒)可用，DSB可以通过同源重组(HR)途径修复，向基因组中插入新的遗传物质。在其他系统中已经表明，当使用单链细小病毒基因组作为具有DSB的同源模板时，每个细胞以10-1到10-3个事件的频率发生位置特异性基因替换-比使用传统的质粒模板的自发HR(无DSB)的效率高达1000万倍。我们实验室拥有唯一已知的能够感染AN的细小病毒。冈比亚按蚊(冈比亚按蚊致密核增多症病毒；AgDNV)，我们相信它将作为一种高效的载体，运送能够精确操纵冈比亚按蚊基因组的试剂。在这项提案中，我们将使用体外系统来研究这个问题。我们的总体假设是，AgDNV可以单独或与同源基因替换盒一起转导特定的ZFN，以实现高效的基因敲除或基因替换。这一假说将通过以下3个具体目标得到验证：1)建立一个基于AgDNV的靶向基因干扰系统，在冈比亚按蚊细胞中转导位点特异的ZFN；2)利用重组的AgDNV供体载体在冈比亚按蚊细胞中通过同源重组进行基因置换；3)检测ZFN介导的位点特异性DSB对AgDNV介导的基因置换的协同作用。 公共卫生相关性：对主要疟疾媒介冈比亚按蚊的基础和应用分子研究面临的最大障碍之一是该物种的转基因效率低得令人望而却步。按蚊研究界将从能够实现有针对性的基因敲除和有针对性的转基因整合的工具中受益匪浅。在这项提案中，我们将开发一个超高效的冈比亚按蚊基因组位点特异性基因敲除和敲入系统。

DESCRIPTION (provided by applicant): One of the greatest obstacles facing basic and applied molecular Anopheles gambiae research is the prohibitively low efficiency of transgenesis for this species. In the twenty years since first report of a stable germline transformation, only a handful of successful transgenic An. gambiae lines have been published. The dearth of tools for targeted gene knockouts and gene replacements in Anopheles gambiae is currently recognized as a critical research need. In other systems, Zinc-Finger Nucleases (ZFNs) are very efficient tools for targeted gene-disruption by inducing a double strand break (DSB). When no homologous donor template is provided, ZFN-induced DSBs result high efficiency gene-disruption through non-homologous end-joining (NHEJ)-mediated frame-shifts. When a DSB occurs, if a homologous template (such as a plasmid) is available the DSB can be repaired by the homologous recombination (HR) pathway, inserting new genetic material into the genome. It has been shown in other systems that when a single-stranded parvovirus genome is used as the homologous template with a DSB, site-specific gene replacement occurs at a frequency of 10-1 to 10-3 events per cell - up to 10 million times greater efficiency than spontaneous HR (no DSB) with a traditional plasmid template. Our laboratory possesses the only known parvovirus capable of infecting An. gambiae (the Anopheles gambiae densonucleosis virus; AgDNV), which we believe will serve as a high-efficiency vector for the delivery of agents capable of precise manipulation of the Anopheles gambiae genome. In this proposal, we will investigate this question using an in vitro system. Our overall hypothesis is that AgDNV can be used to transduce specific ZFNs alone or in conjunction with homologous gene replacement cassettes for high- efficiency gene knockout or gene replacement. This hypothesis will be examined by the following 3 specific Aims: 1) establish a system for targeted gene-disruption by AgDNV-based transduction of site-specific ZFNs in Anopheles gambiae cells, 2) use recombinant AgDNV donor vectors for gene replacement by homologous recombination in Anopheles gambiae cells and 3) examine the synergistic effect of ZFN-mediated site-specific DSBs on AgDNV-mediated gene-replacement. PUBLIC HEALTH RELEVANCE: One of the greatest obstacles facing basic and applied molecular research on the major malaria vector Anopheles gambiae is the prohibitively low efficiency of transgenesis for this species. The Anopheles research community will benefit tremendously from tools that enable targeted gene knock-outs and targeted integration of transgenes. In this proposal, we will develop a system for ultra- high efficiency site-specific gene knock-outs and knock-ins for the Anopheles gambiae genome.