Chimeric Nucleases for Gene Targeting in Drosophila

描述（由申请人提供）：基因靶向涉及同源 在一些实施方案中，所述方法包括引入的DNA与染色体基因座之间的重组。效率 这一进程的一般是相当低的，它似乎是有限的， 染色体目标的状态。这项提案的目的是测试 这些试剂有望通过以下方式提高成功基因靶向的频率： 特异性地切割靶DNA。我们正在研究嵌合核酸酶 该联合收割机将由锌指组成的DNA结合结构域与非特异性 DNA切割结构域。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 具有将切割引导至宽范围的靶位点的潜力。虽然 所提出的实验仅限于果蝇，这种基因靶向 该方法具有很强的通用性，适用于动物的繁殖 人类疾病的模型，并最终用于人类基因治疗。 嵌合核酸酶刺激基因靶向的能力将 在果蝇中进行测试，果蝇。一个特定的目标 选择黄色（y）基因，识别该序列的核酸酶具有 已经建成。为了测试这些酶找到并切割它们的能力， 在果蝇基因组中的靶点，核酸酶的表达将被诱导， 幼虫和新出现的成虫筛选新的y突变， 不准确的修复造成的然后将线性供体DNA提供给 在断裂处引入特定的突变。各种递送方法 将比较嵌合核酸酶和供体DNA，以找到具有嵌合核酸酶的方案。 方便与高效的最佳结合。系统参数将 研究，包括同源性要求，大小的影响， 和引入突变的位置，以及禁用DNA的影响， 有助于同源或非同源重组的修复基因 途径。为了测试该方法的通用性，将其应用于第二个 果蝇基因座，rosy（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.