Key result
Computationally designed integrated zinc finger nucleases demonstrated DNA specificity, metal binding, and in situ intramolecular activation resulting in specific cleavage of DNA with moderate activity.
The study demonstrates a new approach to artificial nuclease design using integrated nucleases with regulator, DNA-binding, and nuclease units to potentially improve gene editing safety.
May inform safer gene-editing nuclease design; leaves open in vivo human translation.
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.
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Németh et al. (2017) studied this question. Integrated zinc finger nucleases was evaluated on DNA specificity, metal binding, and specific cleavage of DNA. Computationally designed integrated zinc finger nucleases demonstrated DNA specificity, metal binding, and in situ intramolecular activation resulting in specific cleavage of DNA with moderate activity.
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