Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases
Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases
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DOI：
10.1038/nbt1317
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发表时间：
2007-07-01
影响因子：
46.9
通讯作者：
Cathomen, Toni
中科院分区：
文献类型：
--
作者：
Szczepek, Michal;Brondani, Vincent;Cathomen, Toni
由拴系到工程化锌指DNA结合蛋白的FokI切割结构域组成的人工核酸内切酶已被证明可用于刺激多种细胞类型中的同源重组。因为锌指核酸酶（ZFN）的催化结构域必须二聚化以变得有活性，所以两个亚基通常在切割位点处组装为异二聚体。ZFN的使用通常与显著的细胞毒性相关，推测是由于在脱靶位点的切割。在这里，我们描述了一种基于结构的方法来减少脱靶切割。使用计算机蛋白质建模和能量计算，我们通过防止同源二聚化和降低二聚化能量来增加靶位点切割的特异性。基于细胞的重组试验证实，修饰的ZFN与原始ZFN一样具有活性，但引起的遗传毒性显著降低。改善的安全性特征可以促进ZFN技术的治疗应用。
Artificial endonucleases consisting of a Fokl cleavage domain tethered to engineered zinc-finger DNA-binding proteins have proven useful for stimulating homologous recombination in a variety of cell types. Because the catalytic domain of zinc-finger nucleases (ZFNs) must dimerize to become active, two subunits are typically assembled as heterodimers at the cleavage site. The use of ZFNs is often associated with significant cytotoxicity, presumably due to cleavage at off- target sites. Here we describe a structure- based approach to reducing off- target cleavage. Using in silico protein modeling and energy calculations, we increased the specificity of target site cleavage by preventing homodimerization and lowering the dimerization energy. Cell-based recombination assays confirmed that the modified ZFNs were as active as the original ZFNs but elicit significantly less genotoxicity. The improved safety profile may facilitate therapeutic application of the ZFN technology.