Chemical Approach to Biological Safety: Molecular-Level Control of an Integrated Zinc Finger Nuclease
| Autor: | Béla Gyurcsik, Hans Erik Mølager Christensen, Chris Oostenbrink, Kohsuke Kato, Masamitsu N. Asaka, Eszter Németh, Zita Fábián, Kyosuke Nagata |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Spectrometry Mass Electrospray Ionization Electrophoretic Mobility Shift Assay Cleavage (embryo) Biochemistry 03 medical and health sciences chemistry.chemical_compound Genome editing Catalytic Domain Humans Molecular Biology Zinc finger Nuclease Transcription activator-like effector nuclease biology Circular Dichroism Organic Chemistry DNA Zinc finger nuclease Zinc Finger Nucleases Kinetics HEK293 Cells 030104 developmental biology Microscopy Fluorescence chemistry Metals Colicin biology.protein Molecular Medicine |
| Zdroj: | ChemBioChem |
| ISSN: | 1439-4227 |
| DOI: | 10.1002/cbic.201700420 |
| Popis: | Application of artificial nucleases (ANs) in genome editing is still hindered by their cytotoxicity, related to off-target cleavages. This problem could be targeted by the regulation of the nuclease domain. Here we provide the experimental survey of computationally designed integrated zinc finger nucleases, constructed by linking the inactivated catalytic centre and the allosteric activator sequence of the colicin E7 nuclease domain to the two opposite termini of a zinc finger array. DNA-specificity and metal binding were confirmed by electrophoretic mobility shift assays, synchrotron radiation circular dichroism spectroscopy and nano-electrospray ionization mass spectrometry. In situ intramolecular activation of the nuclease domain was observed resulting in specific cleavage of DNA with moderate activity. This study represents a new approach to AN design through integrated nucleases consisting of three (regulator - DNA-binding - nuclease) units rather than simple chimera. The optimization of such ANs can lead us to safe gene editing enzymes. |
| Databáze: | OpenAIRE |
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