Chemical Approach to Biological Safety Molecular-Level Control of an Integrated Zinc Finger Nuclease /

Application of artificial nucleases (ANs) in genome editing is still hindered by their cytotoxicity related to off-target cleavages. This problem can be targeted by regulation of the nuclease domain. Here, we provide an experimental survey of computationally designed integrated zinc finger nucleases...

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Bibliographic Details
Main Authors: Németh Eszter
Asaka MN Masamitsu
Kato Kohsuke
Fabian Zita
Oostenbrink Chris
Christensen Hans
Nagata Kyosuke
Gyurcsik Béla
Format: Article
Published: 2018
Series:CHEMBIOCHEM 19 No. 1
doi:10.1002/cbic.201700420

mtmt:3322174
Online Access:http://publicatio.bibl.u-szeged.hu/16376
Description
Summary:Application of artificial nucleases (ANs) in genome editing is still hindered by their cytotoxicity related to off-target cleavages. This problem can be targeted by regulation of the nuclease domain. Here, we provide an experimental survey of computationally designed integrated zinc finger nucleases, constructed by linking the inactivated catalytic centre and the allosteric activator sequence of the colicinE7 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 ionisation 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, and nuclease) units, rather than simple chimera. The optimisation of such ANs could lead to safe gene editing enzymes.
Physical Description:66-75
ISSN:1439-4227