DNA-binding Specificity Is a Major Determinant of the Activity and Toxicity of Zinc-finger Nucleases

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 do...

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Published inMolecular therapy Vol. 16; no. 2; p. 352
Main Authors Cornu, Tatjana I, Thibodeau-Beganny, Stacey, Guhl, Eva, Alwin, Stephen, Eichtinger, Magdalena, Joung, J K, Cathomen, Toni
Format Journal Article
LanguageEnglish
Published United States 01.02.2008
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Summary: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.
ISSN:1525-0024