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Mol Ther. 2008 Feb;16(2):352-8. Epub 2007 Nov 20.

DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases.

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Charité Medical School, Institute of Virology (CBF), Berlin, Germany.

Erratum in

  • Mol Ther. 2008 Nov;16(11):1898. Joung, J K [corrected to Joung, J Keith].


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.

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