Design of Theophylline Binding Guide RNA for Allosteric Regulation of CRISPR/Cas9 System

The possibility of CRISPR/Cas gene editing system regulation is an important point for creation of effective molecular biological instrument. Allosteric regulation at the level of guide RNAs is one of possible approach to regulate gene editing system. In this work, we designed chimeric guide RNAs (sgRNA, crRNA, and tracrRNA) containing an aptamer to theophylline in their structure in order to create gene editing systems whose activity can be regulated (increased or decreased) allosterically. Two approaches were used to design the guide RNAs. In the first variant, aptamer sequence was a part of the main elements of guide RNAs. In the second variant, the aptamer sequence was added to the 3'- or 5'-end of the guide RNAs. Changes in Cas9 nuclease activity in the presence of constructed chimeric guide RNAs and the effect of theophylline on the efficiency of model DNA cleavage were studied. Several promising candidates for the role of components of the theophylline-activated and deactivated CRISPR/Cas9 system have been discovered.