Journal
JOURNAL OF BIOTECHNOLOGY
Volume 140, Issue 3-4, Pages 156-161Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2009.02.004
Keywords
Artificial zinc-finger protein; DNA cleavage; Protein engineering; Zinc-finger nuclease
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Funding
- Japan Society for the Promotion of Science [17550154]
- Grants-in-Aid for Scientific Research [17550154] Funding Source: KAKEN
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Zinc-finger-FokI nucleases (ZFNs) are useful for manipulating genomic DNA, but two ZFNs are required to cleave one site of double-stranded DNA (dsDNA), which limits the choice of targets. To refine ZFN technology, we constructed artificial zinc-finger nucleases containing an artificial zinc-finger protein (AZP) and a single-chain FokI dimer with nine different peptide linkers between two FokI molecules (designated AZP-scFokI). DNA cleavage assays revealed that the AZP-scFokI variant possessing the longest peptide linker cleaved dsDNA with equal or greater reactivity than the corresponding AZP-FokI dimer. The DNA cleavage pattern of AZP-scFokI suggests that the enhanced dsDNA cleavage was due to increased formation of FokI dimer in AZP-scFokI. Furthermore, we demonstrated that AZP-scFokI site-specifically cleaved its target DNA due to the AZP moiety discriminating one base pair difference. Thus, a single AZP-scFokI molecule is able to cleave dsDNA efficiently and site-specifically, and enhances the usefulness of the ZFN approach. (C) 2009 Elsevier B.V. All rights reserved.
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