Research abstract
Article abstract
Nature Biotechnology 25, 786 - 793 (2007)
Published online: 1 July 2007 | doi:10.1038/nbt1317
Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases
Michal Szczepek1,4, Vincent Brondani2,4, Janine Büchel1,4, Luis Serrano3, David J Segal2 & Toni Cathomen1
Abstract
Artificial endonucleases consisting of a FokI 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.
- Charité Medical School, Institute of Virology (CBF), 12203 Berlin, Germany.
- University of California Davis, Genome Center and Department of Pharmacology, Davis, California 95616, USA.
- Centre de Regulacio Genomica, CRG-EMBL Systems Biology Unit, 08003 Barcelona, Spain.
- These authors contributed equally to this work.
Correspondence to: Toni Cathomen1 e-mail: toni.cathomen@charite.de
Correspondence to: David J Segal2 e-mail: djsegal@ucdavis.edu
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