Sandwiched zinc-finger nucleases harboring a single-chain FokI dimer as a DNA-cleavage domain.
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.
Mori
Tomoaki
T
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyotodaigaku-Katsura, Kyoto 615-8510, Japan.
Kagatsume
Ikuko
I
Shinomiya
Kazuki
K
Aoyama
Yasuhiro
Y
Sera
Takashi
T
D004247:DNA / Q000737:chemistry*; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D055503:Protein Multimerization; D016335:Zinc Fingers
D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific