Custom zinc-finger nucleases for use in human cells
Custom zinc-finger nucleases for use in human cells
复制标题
DOI：
10.1016/j.ymthe.2005.06.094
复制
发表时间：
2005-10-01
影响因子：
12.4
通讯作者：
Cathomen, T
中科院分区：
文献类型：
--
作者：
Alwin, S;Gere, MB;Cathomen, T
通过同源重组（HR）进行基因组工程是研究生物学途径或为遗传疾病创造治疗方案的有力工具。在哺乳动物细胞中，HR是罕见的，但靶向DNA双链断裂的产生显著刺激HR。在这里，我们提出了一种方法来产生，评估和优化合理设计的核酸内切酶，促进HR。DNA结合域通过组装预定义的锌指模块选择噬菌体展示合成。转录激活结构域的连接允许在报告基因测定中评估DNA结合，而与核酸内切酶结构域的融合产生定制核酸酶，在附加型和染色体基因修复测定中测试其刺激HR的能力。我们证明了特异性，表达动力学和蛋白质设计是有效基因修复的关键参数，并且我们的两步测定允许人们快速地从设计到测试，再到在人类细胞中成功使用定制核酸酶。
Genome engineering through homologous recombination (HR) is a powerful instrument for studying biological pathways or creating treatment options for genetic disorders. In mammalian cells HR is rare but the creation of targeted DNA double-strand breaks stimulates HR significantly. Here, we present a method to generate, evaluate, and optimize rationally designed endonucleases that promote HR. The DNA-binding domains were synthesized by assembling predefined zinc-finger modules selected by phage display. Attachment of a transcriptional activation domain allowed assessment of DNA binding in reporter assays, while fusion with an endonuclease domain created custom nucleases that were tested for their ability to stimulate HR in episomal and chromosomal gene repair assays. We demonstrate that specificity, expression kinetics, and protein design are crucial parameters for efficient gene repair and that our two-step assay allows one to go quickly from design to testing to successful employment of the custom nucleases in human cells.