Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer

Takashi Mino, Yasuhiro Aoyama, Takashi Sera

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)156-161
Number of pages6
JournalJournal of Biotechnology
Volume140
Issue number3-4
DOIs
Publication statusPublished - Mar 25 2009
Externally publishedYes

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Keywords

  • Artificial zinc-finger protein
  • DNA cleavage
  • Protein engineering
  • Zinc-finger nuclease

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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