Gene Targeting in Flies with ZFNs

描述（由申请人提供）：锌指核酸酶（ZFNs）正在成为许多生物靶向基因修饰的强大工具。在特定基因中进行这种改变的能力有助于分析基因功能，构建人类疾病的实验模型，并最终为人类基因治疗提供疾病基因的定向改变。目前的提案解决了几个问题，这些问题涉及到ZFNs应用的最佳途径，使用果蝇，黑腹果蝇，作为实验生物。1)将测试许多参数对基因靶向效率的影响，包括所需的同源性数量以及在供体DNA中包含相当大的插入和缺失的影响。2)新锌指套设计的几个方面将通过将该技术应用于几个新的果蝇基因来进行测试。3)将进行广泛的深度测序研究，以确定除了设计的靶标之外，基因组中哪些特定的zfn对会导致DNA断裂。这些脱靶切割是毒性的来源，已经在许多ZFN结构体中检测到。对这些位点的位置有一个清晰的了解，将有助于推导体内锌指识别的规则，并有助于检测由于ZFN切割引起的不良影响。4)单链断裂刺激基因靶向的能力将被测试。如前所述，由规范zfn产生的双链断裂可能具有有害的影响。单链断裂可能更安全，如果它们能在合理水平上刺激靶向，而不通过双链断裂的中间过程。所有这些研究的结果将直接关系到ZFN在其他生物中的靶向，包括人类细胞。

DESCRIPTION (provided by applicant): Zinc-finger nucleases (ZFNs) are emerging as powerful tools for targeted gene modification in many organisms. The ability to make such changes in specific genes facilitates the analysis of gene function, the construction of experimental models of human disease, and ultimately the directed alteration of disease genes for human gene therapy. The current proposal addresses several questions that concern the best approach to these applications of ZFNs, using the fruit fly, Drosophila melanogaster, as the experimental organism. 1) A number of parameters will be tested for their effects on the efficiency of gene targeting, including the amount of homology required and the effects of including rather large insertions and deletions in the donor DNA. 2) Several aspects of the design of new zinc finger sets will be tested by application of the technology to several new Drosophila genes. 3) An extensive, deep-sequencing study will be done to determine where in the genome, in addition to the designed target, specific pairs of ZFNs make DNA breaks. These off-target cuts are the source of toxicity that has been detected for a number of ZFN constructs. Having a clear picture of where these sites are will allow derivation of rules regarding zinc-finger recognition in vivo and will facilitate detection of undesirable effects due to ZFN cleavage. 4) The ability of single-strand breaks to stimulate gene targeting will be tested. The double-strand breaks created by canonical ZFNs can have deleterious effects, as noted. Single-strand breaks may be safer, if they can stimulate targeting at reasonable levels without proceeding through double-strand-break intermediates. The results of all these studies will be directly relevant to ZFN targeting in other organisms, including human cells. PUBLIC HEALTH RELEVANCE: The results of this study will guide research into fundamental gene function, including genes of relevance to human health. They will provide information on the best methods for creating models of human disease in experimental organisms and for modifying genes in applications to human gene therapy.