Use of RecA to Promote Gene Targeting

描述（由申请人提供）：在斑马鱼中进行的前瞻性基因筛选已经牢固地建立了该系统作为人类疾病和发展的模型。斑马鱼基因组测序和强大的基于基因组学的工具的发展也有助于将该模型系统置于脊椎动物发育遗传学的前沿。尽管斑马鱼具有这种突出的作用，但在该模型系统中充分利用基于基因组学的工具的能力受到无法进行反向遗传学的限制。为了满足这一需求，我们建议开发简单的方法，大多数具有中等分子生物学技能的实验室都可以使用这些方法来破坏或靶向基因。我们的方法使用了细菌重组酶蛋白RecA的显著修改版本，其活性大大增强。RecA是一种参与重组和修复双链断裂的多功能酶。RecA具有多种酶功能，包括包裹单链DNA，进行同源性搜索，以及在重组过程中促进链交换的能力。我们建议使用我们改良的RecA包覆单链DNA，并产生与我们希望靶向的基因同源的DNA-RecA细丝。在Aim 1中，我们将测试这些细丝是否可以在斑马鱼基因组中产生小的缺失和插入。在Aim 2中，我们将确定单链DNA-RecA细丝促进报告基因靶向插入斑马鱼基因组的频率。上述两个目标中描述的两种活动都与单链DNA-RecA细丝产生双链断裂的能力相一致。我们提供了重要的初步结果来支持这两个目标，并展示了注射嵌合版本的RecA蛋白如何介导斑马鱼早期胚胎有丝分裂细胞和斑马鱼种系中的基因靶向。虽然这些技术正在斑马鱼中开发，但我们相信它们可以适用于大多数人类疾病模型的基因组修饰，并将应用于人类基因治疗。

DESCRIPTION (provided by applicant): The forward genetic screens conducted in zebrafish have firmly established this system as a model for human disease and development. Sequencing of the zebrafish genome and the development of powerful genomics-based tools have also contributed to the placement of this model system at the forefront of vertebrate developmental genetics. Despite this prominent role for zebrafish, the ability to fully utilize genomics- based tools in this model system is limited by the inability to perform reverse genetics. To address this need, we propose to develop simple methods that most labs with moderate molecular biology skills could use to disrupt or target genes. Our method uses a significantly modified version of the bacterial recombinase protein RecA that has greatly enhanced activities. RecA is a multifunctional enzyme involved in recombination and the repair of double-strand breaks. RecA has diverse enzymatic functions, including the ability to coat single-stranded DNA, to carry out homology searches, and to promote strand exchange during recombination. We propose to use our modified version of RecA to coat single stranded DNA and produce DNA-RecA filaments that are homologous to the gene we wish to target. In Aim 1, we will test whether these filaments can create small deletions and insertions in the zebrafish genome. In Aim 2, we will determine the frequency with which the single-stranded DNA-RecA filaments promote targeted insertion of a reporter gene into the zebrafish genome. Both of the activities described in the above two aims would be consistent with the ability of the single- stranded DNA-RecA filaments to create double strand breaks. We provide significant preliminary results to support both of these aims and demonstrate how injection of chimeric versions of RecA proteins can mediate gene targeting in mitotic cells of the early zebrafish embryo and in the zebrafish germline. While these techniques are being developed in zebrafish, we believe they can be adapted for genome modifications in most models for human disease and will have applications for human gene therapy. PUBLIC HEALTH RELEVANCE: We propose to develop methods in zebrafish that utilize the bacterial recombinase protein RecA to create site directed modification of the genome. These techniques have the potential to revolutionize reverse genetic approaches in all animal models of human disease. Successful completion of this proposal will have a profound impact on the identification of pharmaceutical targets for drug discovery and the production of new animal models of human disease.