Sandwiched zinc-finger nucleases demonstrating higher homologous recombination rates than conventional zinc-finger nucleases in mammalian cells |
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| Institution: | 1. Institute for Genetic Medicine, Hokkaido University, Kita-ku, Sapporo 060-0815, Japan;2. DNAVEC Corporation, Techno Park Oho, 6 Ohkubo, Tsukuba, Ibaraki 300-2611, Japan;1. Epidemic Intelligence Service Program, The Office of Surveillance, Epidemiology, and Laboratory Services, Centers for Disease Control, Atlanta, GA, United States;2. Division of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, GA, United States;3. Virginia Department of Health, Richmond, VA, United States;1. Central Institute of Mathematics and Economics, Russian Academy of Sciences, Nakhimovskii prospect 47, 117418 Moscow, Russia;2. CentER, Department of Econometrics and Operations Research, Tilburg University, P.O. Box 90153, 5000 LE Tilburg, The Netherlands;1. Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA;2. Department of Chemistry, Southern Illinois University Edwardsville, IL 62026, USA;1. Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa 761-0795, Japan;2. Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Nakorn Pathom 73140, Thailand;3. Faculty of Agriculture, Chiang Mai University, 50200 Chiang Mai, Thailand;4. Faculty of Biology, Hanoi National University of Education, 136 Xuan Thuy Road, Hanoi, Vietnam;1. School of Electrical Engineering, VIT University, Vellore 632014, Tamil Nadu, India;2. School of Electronics Engineering, VIT University, Vellore 632014, Tamil Nadu, India;3. Sankalp and KPIT Semiconductors Pvt. Ltd, Bangalore, Karnataka, India;4. Department of ECE, KCG College of Technology, Chennai 600097, Tamil Nadu, India |
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| 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. |
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| Keywords: | Sandwiched zinc finger nuclease Single-chain FokI dimer Zinc finger protein Homologous recombination Genome editing |
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