• CRISPR/Cas
• gRNA
• nucleic acids
• Cas12a
• DNA
• fluorescence signal amplification.

In this thesis a DNA-based circuit was developed and subsequently characterized with the aim of obtaining an exponential amplification of the fluorescence signal in biosensing platforms based on CRISPR/Cas12a.
The binding between gRNA and ssDNA was studied by FRET technique at two different temperatures (25.0 and 37.0°C). The thermodynamic and kinetic constants have been estimated and two-step reaction mechanism was hypothesised. The results, albeit with large uncertainties, have found support in the literature. As far as we know, this is the first attempt to study binding between a gRNA and a ssDNA. The trans-activity of the Cas12a/gRNA complex (E. coli) was then studied both on the linear reporter (5 nt) and on a hairpin reporter. The efficiency of the two reporters (kcat/KM ratio) was similar. The cutting mechanism of the hairpin reporter was investigated with the PAGE technique, but without providing very useful results. Finally, we were able to perform a qualitative amplification experiment by selecting the concentrations of all strands based on the kcat and K values obtained in the previous characterisation work. The first results were unfortunately not very encouraging, likely due either to the cutting mechanism of the hairpin reporter or to the binding between gRNA and ssDNA (the presence of the enzyme was neglected).