Chemical Approach to Biological Safety: Molecular‐Level Control of an Integrated Zinc Finger Nuclease. (12th December 2017)

• Record Type: Journal Article
• Authors: Németh, Eszter; Asaka, Masamitsu N.; Kato, Kohsuke; Fábián, Zita; Oostenbrink, Chris; Christensen, Hans E. M.; Nagata, Kyosuke; Gyurcsik, Béla
• Is Part Of: Chembiochem. Volume 19:Number 1(2018)
• Page Start: 66
• Page End: 75
• Publication Date: 2017-12-12
• Subjects: allosteric activation -- colicin E7 -- DNA nicking -- enzyme design -- zinc finger nuclease; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201700420
• Languages: English
• ISSNs: 1439-4227

Abstract

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. Abstract : Intelligently artificial : We constructed integrated artificial nucleases from an inactivated nuclease domain to avoid side effects upon partial degradation. The catalytic reaction was induced by specific DNA binding of the zinc fingers and by the third unit of the enzyme, the N‐terminal regulatory segment. The computationally designed colicin E7 nuclease domain‐based enzymes performed specific nicking of DNA.