Efficient site-specific genetic engineering of the Anopheles gambiae genome

描述（由申请人提供）：冈比亚按蚊分子基础和应用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。自首次报道稳定的种系转化以来的20年里，只有少数转基因水稻获得成功。冈比亚的线条已经出版。缺乏针对冈比亚按蚊的靶向基因敲除和基因替换的工具目前被认为是一个关键的研究需求。在其他系统中，锌指核酸酶（ZFNs）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。当没有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率的基因破坏。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带有DSB的同源模板时，每个细胞发生位点特异性基因替换的频率为10-1至10-3次，比使用传统质粒模板的自发HR（无DSB）效率高出1000万倍。我们实验室有唯一已知的能感染安的细小病毒。冈比亚疟蚊（冈比亚按蚊致密核病病毒；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药物。在本提案中，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可用于单独转导特定的ZFNs或与同源基因替换盒结合，以实现高效的基因敲除或基因替换。这一假设将通过以下3个具体目标进行验证：1)建立基于AgDNV的冈比亚按蚊细胞中位点特异性ZFNs转导的靶向基因破坏系统；2)利用重组AgDNV供体载体在冈比亚按蚊细胞中通过同源重组进行基因替代；3)检验zfn介导的位点特异性DSBs对AgDNV介导的基因替代的协同作用。

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.