Abstract

The emergence of the CRISPR/Cas editing system has already revolutionised modern genetic manipulation, notwithstanding, the application of this technology for in vivo disease treatment is still hampered, with one of the main concerns being specificity and safety. Furthermore, targeted delivery of the CRISPR/Cas system by canonical approaches in organisms is challenging, mostly due to ineffectiveness and the establishment of adverse side-effects, demanding thus for means allowing to confine this technology in space and time.

To overcome these limitations, we propose the rational design of a remotely-controllable and non-invasive nanotransducer by combining the field of synthetic biology with nanotechnology. Splitting an enzyme into two portions to abolish its activity represents a powerful strategy for its regulation, and controlling the reconstitution of the single fragments with tuneable modules adds an additional layer of control. In this framework, we focussed first on the development of a Split Cas9 equipped with photoactivatable domains which enable allosteric activation only upon conditional heterodimerization. The final design envisages further the conjugation of a gold nanoparticle (AuNP) to the recombinant proteins. Despite enhancing protein stability and favouring cellular delivery, AuNPs produce highly localized heat under illumination at their plasmonic resonance wavelength. Property that is exploitable to disengage enzyme activity through thermal protein denaturation, further limiting the tempo-spatial activity window of our device. After successful genetic engineering of the Split Cas9 portions, we confirmed the conditional reconstitution of the complex upon blue-light exposure. Moreover, preliminary data showed gene editing activity in vivo, as recombinant proteins-based system were validated in zebrafish embryos. Subsequently, we evaluated the efficiency of inducible gene editing in melanoma cells. Ultimately, the cellular uptake of the conjugated Split Cas9 protein and the intracellular assembly of the complex has been assessed. The development of the here presented nanotransducer provides thus the basis for designing programmable biological systems that can be exploited in manipulating cellular processes at distance, while ensuring stable control at a high resolution. The ability to selectively activate biological events in a predictable manner carries a huge benefit, not only for basic research, but it provides a potential tool for future applications in personalized nanomedicine and biotechnology.