PMID:18026168
| Citation |
Cornu, TI, Thibodeau-Beganny, S, Guhl, E, Alwin, S, Eichtinger, M, Joung, JK, Joung, JK and Cathomen, T (2008) DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases. Mol. Ther. 16:352-8 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1038/sj.mt.6300357 |
| Keywords |
Amino Acid Sequence; Cell Line; DNA/metabolism; Endonucleases/genetics; Endonucleases/metabolism; Flow Cytometry; Genetic Vectors/genetics; Humans; Immunoblotting; Molecular Sequence Data; Plasmids/genetics; Protein Binding; Recombination, Genetic; Retroviridae/genetics; Sequence Homology, Amino Acid; Transcription, Genetic; Zinc Fingers |
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