1. ¹Charité Medical School, Institute of Virology (CBF), Berlin, Germany
2. ²Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital,Charlestown,Massachusetts,USA
3. ³Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

Correspondence: Toni Cathomen, Charité Medical School, Institute of Virology (CBF), Hindenburgdamm 27, D-12203 Berlin, Germany. E-mailtoni.cathomen@charite.de

Received 1 September 2007; Accepted 15 October 2007; Published online 20 November 2007.

Abstract

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.