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 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.
Charité Medical School, Institute of Virology (CBF), Berlin, Germany.
Tatjana I
TI
Cornu
Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
Stacey
S
Thibodeau-Beganny
Charité Medical School, Institute of Virology (CBF), Berlin, Germany.
Eva
E
Guhl
Charité Medical School, Institute of Virology (CBF), Berlin, Germany.
Stephen
S
Alwin
Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA; Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
Magdalena
M
Eichtinger
Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA; Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
J K
JK
Joung
Charité Medical School, Institute of Virology (CBF), Berlin, Germany. Electronic address: toni.cathomen@charite.de.
Toni
T
Cathomen