title: DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases.
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.
authors.affiliation: Charité Medical School, Institute of Virology (CBF), Berlin, Germany.
authors.forename: Tatjana I
authors.initials: TI
authors.lastname: Cornu
authors.forename: Stacey
authors.initials: S
authors.lastname: Thibodeau-Beganny
authors.forename: Eva
authors.initials: E
authors.lastname: Guhl
authors.forename: Stephen
authors.initials: S
authors.lastname: Alwin
authors.forename: Magdalena
authors.initials: M
authors.lastname: Eichtinger
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
authors.forename: J K
authors.initials: JK
authors.lastname: Joung
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D016335:Zinc Fingers
chemical_list: D004247:DNA; D004720:Endonucleases