TY - JOUR
T1 - Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer
AU - Mino, Takashi
AU - Aoyama, Yasuhiro
AU - Sera, Takashi
N1 - Funding Information:
We thank Haruyuki Atomi for the use of their DNA sequencer. This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 17550154 to T.S.).
PY - 2009/3/25
Y1 - 2009/3/25
N2 - 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.
AB - 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.
KW - Artificial zinc-finger protein
KW - DNA cleavage
KW - Protein engineering
KW - Zinc-finger nuclease
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U2 - 10.1016/j.jbiotec.2009.02.004
DO - 10.1016/j.jbiotec.2009.02.004
M3 - Article
C2 - 19428709
AN - SCOPUS:62849090902
VL - 140
SP - 156
EP - 161
JO - Journal of Biotechnology
JF - Journal of Biotechnology
SN - 0168-1656
IS - 3-4
ER -