Chimeric Nucleases for Gene Targeting in Drosophila

描述(申请人提供)：基因打靶涉及同源基因 引入的DNA和染色体基因座之间的重组。效率 这一过程的效率通常很低，而且似乎受到 染色体靶标的状态。这项提议的目的是为了测试 有望通过以下方式提高基因打靶成功率的试剂 特别是切割目标DNA。我们正在研究嵌合核酸酶 它结合了由锌指组成的DNA结合域和非特异性 DNA裂解结构域。因为锌指识别的DNA序列可以 通过改变蛋白质中的几个关键氨基酸来改变，这些嵌合体 有可能直接切割到广泛的靶点。虽然 拟议的实验仅限于果蝇，这种基因靶向 方法是非常普遍的，应该适用于动物的繁殖 人类疾病的模型，并最终应用于人类的基因治疗。 嵌合核酸酶刺激基因靶向的能力将 在果蝇身上进行测试，黑腹果蝇。一个特定的目标一直是 在黄色(Y)基因中选择，识别该序列的核酸酶具有 已经建造好了。为了测试这些酶发现和切割它们的能力 在果蝇基因组中的靶标，核酸酶的表达将在 幼虫和初露头角的成虫筛选发生的新的y突变 通过不准确地修复断口。然后线性供体DNA将被提供给 在休息时引入一种特定的突变。交付的各种方法 嵌合核酸酶和供体DNA将进行比较，以找到与 便利性和效率的最佳结合。系统的参数将 被调查，包括同源性要求，大小的影响 以及引入的突变的位置，以及使DNA失效的影响 修复有助于同源或非同源重组的基因 小路。为了测试该方法的通用性，它将应用于第二个 果蝇基因，玫瑰色(Ry)基因。

DESCRIPTION (provided by applicant): Gene targeting involves homologous recombination between an introduced DNA and a chromosomal locus. The efficiency of this process is generally quite low, and it appears to be limited by the status of the chromosomal target. The purpose of this proposal is to test reagents that promise to elevate the frequency of successful gene targeting by specifically cleaving the target DNA. We are working with chimeric nucleases that combine a DNA-binding domain comprised of zinc fingers with a nonspecific DNA-cleavage domain. Because the DNA sequences recognized by zinc fingers can be altered by changing a few key amino acids in the protein, these chimeras have the potential to direct cleavage to a wide range of target sites. Although the proposed experiments are restricted to Drosophila, this gene targeting method is very general and should be applicable to the generation of animal models of human disease and ultimately to human gene therapy. The capabilities of the chimeric nucleases for stimulating gene targeting will be tested in the fruit fly, Drosophila melanogaster. A specific target has been chosen in the yellow (y) gene, and nucleases that recognize that sequence have been constructed. To test the ability of these enzymes to find and cleave their target in the Drosophila genome, expression of the nucleases will be induced in larvae, and the emerging adults screened for new y mutations that have occurred by inaccurate repair of the break. Then a linear donor DNA will be provided to introduce a specific mutation at the break. Various methods of delivering the chimeric nucleases and donor DNA will be compared to find the protocol with the best combination of convenience and efficiency. Parameters of the system will be investigated, including the homology requirements, the effects of the size and location of the introduced mutation, and the effects of disabling DNA repair genes that contribute to homologous or nonhomologous recombination pathways. To test the generality of the method, it will be applied to a second Drosophila locus, the rosy (ry) gene.