Gene Targeting in Flies with ZFNs

描述(申请人提供)：锌指核酸酶(ZFN)正在成为许多生物体中靶向基因修饰的强大工具。在特定基因上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定向改变疾病基因。目前的提案解决了几个涉及ZFN应用的最佳方法的问题，使用果蝇作为实验有机体。1)将测试一些参数对基因打靶效率的影响，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)新的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测试。3)将进行广泛的深度测序研究，以确定除了设计的靶点外，特定的ZFN对在基因组中的哪里造成DNA断裂。这些非靶标切割是已检测到的许多ZFN结构的毒性来源。清楚地了解这些位点的位置将允许推导出关于体内锌指识别的规则，并将有助于检测由于ZFN切割而产生的不良影响。4)将测试单链断裂刺激基因靶向的能力。正如所指出的那样，规范的ZFN造成的双链断裂可能会产生有害的影响。单链断裂可能更安全，如果它们能够在合理水平刺激靶向，而不通过双链断裂中间体。所有这些研究的结果都将与ZFN在包括人类细胞在内的其他生物体中的靶向直接相关。 公共卫生相关性：这项研究的结果将指导对基本基因功能的研究，包括与人类健康相关的基因。他们将提供关于在实验生物体中建立人类疾病模型和在人类基因治疗应用中修改基因的最佳方法的信息。

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.