CHIMERIC NUCLEASES FOR GENE TARGETING

基因打靶的效率受到限制，至少在一定程度上是因为染色体靶标无法进入细胞重组机制。对多种生物体的研究表明，有意切割靶标可以极大地刺激所需的同源重组事件。目前，还没有一种试剂可以有效地在任意选择的靶点上引入双链断裂。这项拟议研究的目标是测试一类新型设计的核酸酶的能力，这些核酸酶很有希望成为靶向重组的启动者。这些酶是FokI限制性内切酶的非特异性DNA裂解结构域和Cys2His2锌指家族的DNA识别结构域之间的工程杂交。这些嵌合核酸酶已被证明在体外直接切割到锌指识别的位置。由于锌指DNA结合域可以被设计或选择来识别各种各样的DNA序列，这些嵌合体应该允许切割指向许多不同的目标。由此产生的程序将立即适用于定向基因操作，用于基因治疗和基因功能的实验研究。拟议研究的最初方面将集中在嵌合核酸酶在体外对目标识别和识别的进一步表征。与体内实验相比，了解酶在没有染色质蛋白质的情况下是如何工作的是重要的，而最佳的识别特异性将是最终基因组切割的关键。嵌合核酸酶的位置将被放置在DNA底物中，以测试非洲爪哇卵母细胞的同源重组能力。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。将对DNA的切割和重组进行评估。非洲爪哇卵母细胞在这些研究中的优势来自于它们大的重组能力，易于底物操作，以及可以表征反应中间产物。一旦确定了嵌合核酸酶的基本性质，将通过随机化和选择技术产生针对新位点的带有锌指的新版本。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.