DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger Nucleases
DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger Nucleases
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DOI：
10.1038/sj.mt.6300357
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发表时间：
2008-02-01
影响因子：
12.4
通讯作者：
Cathomen, Toni
中科院分区：
文献类型：
--
作者：
Cornu, Tatjana I.;Thibodeau-Beganny, Stacey;Cathomen, Toni
操纵细胞基因组的蛋白质工程已发展成为生物医学研究（包括基因治疗）的一项有前途的技术。特别是，锌指核酸酶 (ZFN) 由与定制的锌指 (ZF) DNA 结合域相连的非特异性核酸内切酶域组成，已被证明对于刺激多种细胞类型中的同源定向基因修复具有不可估量的价值。然而，之前的研究表明，由于脱靶位点的裂解，ZFN 可能与显着的细胞毒性相关。在这里，我们将九个 ZF DNA 结合域的体外亲和力和特异性与其在人类细胞中作为 ZFN 的性能进行了比较。我们基于细胞的检测结果表明，除了亲和力之外，DNA 结合特异性是 ZFN 活性的主要决定因素，并且与 ZFN 相关毒性呈负相关。此外，我们的数据提供了第一个证据，证明工程策略（解释上下文相关的 DNA 结合效应）产生的 ZF 在人类细胞中起到高效 ZFN 的作用。
The engineering of proteins to manipulate cellular genomes has developed into a promising technology for biomedical research, including gene therapy. In particular, zinc-finger nucleases (ZFNs), which consist of a nonspecific endonuclease domain tethered to a tailored zinc-finger (ZF) DNA-binding domain, have proven invaluable for stimulating homology-directed gene repair in a variety of cell types. However, previous studies demonstrated that ZFNs could be associated with significant cytotoxicity due to cleavage at off-target sites. Here, we compared the in vitro affinities and specificities of nine ZF DNA-binding domains with their performance as ZFNs in human cells. The results of our cell-based assays reveal that the DNA-binding specificity - in addition to the affinity - is a major determinant of ZFN activity and is inversely correlated with ZFN-associated toxicity. In addition, our data provide the first evidence that engineering strategies, which account for context-dependent DNA-binding effects, yield ZFs that function as highly efficient ZFNs in human cells.