Expanding the Repertoire of Target Sites for Zinc Finger Nuclease-mediated Genome Modification
| Autor: | Kimberly A Wilson, Matthew H. Porteus, Jiuli Zhang, Shondra M. Pruett-Miller, Abbye E. McEwen, Eric J. Kildebeck |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Base pair
genomic modification Computational biology Biology 03 medical and health sciences 0302 clinical medicine Drug Discovery Binding site inter-domain linker 030304 developmental biology Genetics Zinc finger chemistry.chemical_classification 0303 health sciences Nuclease zinc finger lcsh:RM1-950 Zinc finger nuclease zinc finger nuclease Amino acid genomic DNA lcsh:Therapeutics. Pharmacology chemistry biology.protein Molecular Medicine Original Article inter-finger linker Linker 030217 neurology & neurosurgery |
| Zdroj: | Molecular Therapy: Nucleic Acids, Vol 2, Iss C (2013) Molecular Therapy. Nucleic Acids |
| ISSN: | 2162-2531 |
| Popis: | Recent studies have shown that zinc finger nucleases (ZFNs) are powerful reagents for making site-specific genomic modifications. The generic structure of these enzymes includes a ZF DNA-binding domain and nuclease domain (Fn) are separated by an amino acid "linker" and cut genomic DNA at sites that have a generic structure (site1)-(spacer)-(site2) where the "spacer" separates the two binding sites. In this work, we compare the activity of ZFNs with different linkers on target sites with different spacer lengths. We found those nucleases with linkers' lengths of 2 or 4 amino acid (aa) efficiently cut at target sites with 5 or 6 base pair (bp) spacers, and that those ZFNs with a 5-aa linker length efficiently cut target sites with 6 or 7 bp spacers. In addition, we demonstrate that the Oligomerized Pool ENgineering (OPEN) platform used for making three-fingered ZF proteins (ZFPs) can be modified to incorporate modular assembly fingers (including those recognizing ANNs, CNNs, and TNNs) and we were able to generate nucleases that efficiently cut cognate target sites. The ability to use module fingers in the OPEN platform at target sites of 5-7 bp spacer lengths increases the probability of finding a ZFN target site to 1 in 4 bp. These findings significantly expand the range of sites that can be potentially targeted by these custom-engineered proteins.Molecular Therapy - Nucleic Acids (2013) 2, e88; doi:10.1038/mtna.2013.13; published online 30 April 2013. |
| Databáze: | OpenAIRE |
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