Efficient site-specific genetic engineering of the Anopheles gambiae genome

描述（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转基因效率极低。自第一次报道稳定的胚系转化以来的二十年中，只有少数成功的转基因An。冈比亚的路线已经公布。冈比亚按蚊靶向基因敲除和基因置换工具的缺乏是目前公认的一个关键的研究需求。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）介导的移码产生高效的基因破坏。当DSB发生时，如果同源模板（如质粒）可用，则DSB可以通过同源重组（HR）途径修复，将新的遗传物质插入基因组中。在其他系统中已经显示，当单链细小病毒基因组用作具有DSB的同源模板时，位点特异性基因置换以每个细胞10-1至10-3事件的频率发生-比具有传统质粒模板的自发HR（无DSB）的效率高1000万倍。我们的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（冈比亚按蚊浓核病毒; AgDNV），我们相信它将作为一种高效载体，用于递送能够精确操纵冈比亚按蚊基因组的试剂。在这个提议中，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可用于单独或与同源基因置换盒结合以高效率基因敲除或基因置换特异性ZFN。该假设将通过以下3个特定目的进行检验：1）在冈比亚按蚊细胞中建立通过基于AgDNV的位点特异性ZFN转导的靶向基因破坏系统，2）使用重组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，以及3）检查ZFN介导的位点特异性DSB对AgDNV介导的基因置换的协同效应。 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所面临的最大障碍之一是该物种的转基因效率极低。按蚊研究界将极大地受益于工具，使有针对性的基因敲除和有针对性的整合转基因。在本研究中，我们将建立一个冈比亚按蚊基因组的超高效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.