title: Sandwiched zinc-finger nucleases demonstrating higher homologous recombination rates than conventional zinc-finger nucleases in mammalian cells.
abstract: We previously reported that our sandwiched zinc-finger nucleases (ZFNs), in which a DNA cleavage domain is inserted between two artificial zinc-finger proteins, cleave their target DNA much more efficiently than conventional ZFNs in vitro. In the present study, we compared DNA cleaving efficiencies of a sandwiched ZFN with those of its corresponding conventional ZFN in mammalian cells. Using a plasmid-based single-strand annealing reporter assay in HEK293 cells, we confirmed that the sandwiched ZFN induced homologous recombination more efficiently than the conventional ZFN; reporter activation by the sandwiched ZFN was more than eight times that of the conventional one. Western blot analysis showed that the sandwiched ZFN was expressed less frequently than the conventional ZFN, indicating that the greater DNA-cleaving activity of the sandwiched ZFN was not due to higher expression of the sandwiched ZFN. Furthermore, an MTT assay demonstrated that the sandwiched ZFN did not have any significant cytotoxicity under the DNA-cleavage conditions. Thus, because our sandwiched ZFN cleaved more efficiently than its corresponding conventional ZFN in HEK293 cells as well as in vitro, sandwiched ZFNs are expected to serve as an effective molecular tool for genome editing in living cells.
authors.affiliation: Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Mori
authors.affiliation: Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Mori
authors.affiliation: Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Takamasa
authors.initials: T
authors.lastname: Tobimatsu
authors.affiliation: Department of Applied Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan. Electronic address: sera@cc.okayama-u.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sera
mesh_terms: D000818:Animals; D001681:Biological Assay; D002470:Cell Survival / Q000187:drug effects; D053837:DNA Cleavage; D004720:Endonucleases / Q000378:metabolism* / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D057809:HEK293 Cells; D059765:Homologous Recombination; D006801:Humans; D016335:Zinc Fingers / Q000502:physiology
chemical_list: D004720:Endonucleases
keywords: Genome editing; Homologous recombination; Sandwiched zinc finger nuclease; Single-chain FokI dimer; Zinc finger protein