التفاصيل البيبلوغرافية
| العنوان: |
DNA-binding Specificity Is a Major Determinant of the Activity and Toxicity of Zinc-finger Nucleases. |
| المؤلفون: |
Cornu, Tatjana I.1, Thibodeau-Beganny, Stacey2, Guhl, Eva1, Alwin, Stephen1, Eichtinger, Magdalena2,3, Joung, J. K.2,3, Cathomen, Toni1 toni.cathomen@charite.de |
| المصدر: |
Molecular Therapy. Feb2008, Vol. 16 Issue 2, p352-358. 7p. 4 Graphs. |
| مصطلحات موضوعية: |
*DNA-binding proteins, *NUCLEASES, *CELL-mediated cytotoxicity, *ZINC-finger proteins, *GENE therapy, *GENOMES, *HOMOLOGY (Biology), *THERAPEUTICS |
| مستخلص: |
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.Molecular Therapy (2007); 16 2, 352–358. doi:10.1038/sj.mt.6300357 [ABSTRACT FROM AUTHOR] |
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