Sandwiched zinc-finger nucleases harboring a single-chain FokI dimer as a DNA-cleavage domain

Tomoaki Mori, Ikuko Kagatsume, Kazuki Shinomiya, Yasuhiro Aoyama, Takashi Sera

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

Abstract

Zinc-finger nucleases (ZFNs) are a powerful tool for manipulation of genomic DNA. Recently, we reported a new ZFN composed of one artificial zinc-finger protein (AZP) and a single-chain FokI dimer (scFokI) that refines ZFN technology. While AZP-scFokI cleaved DNA specifically around the AZP-target site, several nucleotide positions were cleaved due to the mobility of the scFokI domain. In the present study, we aimed to improve the DNA-cleavage specificity at the nucleotide level. To this end, we sandwiched a scFokI domain between two AZPs to reduce the mobility of the scFokI moiety when bound to DNA. We demonstrated that the AZP-sandwiched scFokI cleaved DNA at a single nucleotide position of a target plasmid, in which two AZP-binding sites were connected with a 6-bp spacer, with multiple turnovers. Further improvement of AZP-sandwiched scFokI will lead to development of ideal artificial meganucleases.

Original languageEnglish
Pages (from-to)694-697
Number of pages4
JournalBiochemical and Biophysical Research Communications
Volume390
Issue number3
DOIs
Publication statusPublished - Dec 18 2009
Externally publishedYes

Keywords

  • Artificial zinc-finger protein
  • Double-stranded DNA cleavage
  • Meganuclease
  • Single-chain FokI dimer
  • Zinc-finger nuclease

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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