CHIMERIC NUCLEASES FOR GENE TARGETING

基因靶向的效率至少部分地受限于染色体靶标对细胞重组机制的不可接近性。 在多种生物体中的研究已经证明，靶的有意切割极大地刺激了期望的同源重组事件。 目前，没有试剂可以有效地在任意选择的靶位点引入双链断裂。 这项研究的目的是测试一类新的设计核酸酶的能力，这些核酸酶作为靶向重组的启动子有很大的希望。 这些酶是FokI限制性内切核酸酶的非特异性DNA切割结构域和Cys2His2锌指家族的DNA识别结构域之间的工程杂交体。 这些嵌合核酸酶已经显示出在体外直接切割到由锌指识别的位点。 由于锌指DNA结合结构域可以被设计或选择为识别各种各样的DNA序列，这些嵌合体应该允许切割被导向许多不同的靶。 由此产生的程序将直接适用于基因治疗和基因功能的实验研究的定向遗传操作。初步方面的拟议研究将集中在进一步表征的目标识别和歧视的嵌合核酸酶在体外。 为了与体内实验进行比较，重要的是要知道在没有染色质蛋白的情况下酶是如何工作的，并且最佳识别特异性对于最终的基因组切割至关重要。 嵌合核酸酶的位点将被放置在DNA底物中，该底物可以测试爪蟾卵母细胞中的同源重组能力。 这些DNA将被引入卵母细胞，在那里它们被组装成染色质，然后注射相应的核酸酶。 将评价DNA的切割和重组。 非洲爪蟾卵母细胞用于这些研究的优势来自于它们的重组能力大，底物操作容易，以及表征反应中间体的可能性。 一旦嵌合核酸酶的基本性质已经确定，将通过随机化和选择技术产生具有靶向新位点的锌指的新版本。 这将为靶向复杂基因组中的特定位点铺平道路。

The efficiency of gene targeting is limited, at least in part, by the inaccessibility of the chromosomal target to cellular recombination machinery. Studies in a variety of organisms have demonstrated that intentional cleavage of the target greatly stimulates the desired homologous recombination event. At present there are no reagents that can efficiently introduce a double-strand break at an arbitrarily chosen target site. The goal of the proposed study is to test the capabilities of a novel class of designed nucleases that have great promise as initiators of targeted recombination. These enzymes are engineered hybrids between the nonspecific DNA cleavage domain of the FokI restriction endonuclease and DNA recognition domains of the Cys2His2 zinc finger family. These chimeric nucleases have been shown to direct cleavage in vitro to sites recognized by the zinc fingers. Because zinc finger DNA-binding domains can be designed or selected to recognize a wide variety of DNA sequences, these chimeras should allow cleavage to be directed to many different targets. The resulting procedures will have immediate applicability to directed genetic manipulations for gene therapy and for experimental investigations of gene function. Initial aspects of the proposed study will focus on further characterization of target recognition and discrimination by the chimeric nucleases in vitro. For comparison with in vivo experiments, it is important to know how the enzymes operate in the absence of chromatin proteins, and optimum recognition specificity will be crucial to ultimate genomic cleavage. Sites for the chimeric nucleases will be placed in DNA substrates that can test homologous recombination capabilities in Xenopus oocytes. These DNAs will be introduced into oocytes, where they are assembled into chromatin, followed by injection of the corresponding nuclease. Both cleavage and recombination of the DNAs will be evaluated. The advantages of Xenopus oocytes for these studies derive from their large capacity for recombination, ease of substrate manipulation, and the possibility of characterizing reaction intermediates. Once the basic properties of the chimeric nucleases have been determined, new versions with zinc fingers targeted to novel sites will be generated by randomization and selection techniques. This will prepare the way for targeting of specific sites in complex genomes.