Chemical Approach to Biological Safety: Molecular-Level Control of an Integrated Zinc Finger Nuclease
2018
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.
Keywords:
- Correction
- Source
- Cite
- Save
- Machine Reading By IdeaReader
66
References
2
Citations
NaN
KQI