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Chemical Approach to Biological Safety: Molecular‐Level Control of an Integrated Zinc Finger Nuclease
Author(s) -
Németh Eszter,
Asaka Masamitsu N.,
Kato Kohsuke,
Fábián Zita,
Oostenbrink Chris,
Christensen Hans E. M.,
Nagata Kyosuke,
Gyurcsik Béla
Publication year - 2018
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201700420
Subject(s) - nuclease , zinc finger , zinc finger nuclease , colicin , transcription activator like effector nuclease , chemistry , dna , genome editing , computational biology , biophysics , combinatorial chemistry , biochemistry , biology , gene , genome , transcription factor , plasmid
Abstract 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 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 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.