title: Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases.
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.
authors.affiliation: Charité Medical School, Institute of Virology (CBF), 12203 Berlin, Germany.
authors.forename: Michal
authors.initials: M
authors.lastname: Szczepek
authors.forename: Vincent
authors.initials: V
authors.lastname: Brondani
authors.forename: Janine
authors.initials: J
authors.lastname: Büchel
authors.forename: Luis
authors.initials: L
authors.lastname: Serrano
authors.forename: David J
authors.initials: DJ
authors.lastname: Segal
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods; D020134:Catalytic Domain; D002460:Cell Line; D045744:Cell Line, Tumor; D018388:Codon, Terminator; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D019281:Dimerization; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D016335:Zinc Fingers
chemical_list: D018388:Codon, Terminator; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific