Multidimensional Control of Cas9 by Evolved RNA Polymerase-Based Biosensors

Systems to control Cas9 with spatial and temporal precision offer opportunities to decrease side effects, protect sensitive tissues, and create gene therapies that are only activated at defined times and places. Here, we present the design of new Cas9 controllers based on RNA polymerase (RNAP)-based biosensors that produce gRNAs, thereby regulating target knockout. After development and validation of a new abscisic acid-inducible biosensor to control Cas9, we lowered the background of the system using continuous evolution. To showcase the versatility of the approach, we designed biosensors that measure medically relevant protein-protein interactions to drive knockout. Finally, to test whether orthogonal RNAP biosensors could integrate multiple input signals to drive multiple gRNA-based outputs with a single Cas9 protein, we designed an on-switch/off switch controller. The addition of one input activates the on switch and induces knockout, while the addition of a second input activates the off switch and produces a gRNA that directs the Cas9 protein to degrade the on switch gRNA vector, thereby deactivating it. This combined activation and deactivation system displayed very low background and inducible target knockout using different combinations of small-molecule treatment. Our results establish engineered RNAP biosensors as deployable Cas9 control elements and open up new opportunities for driving genetic editing technologies by diverse input signals.