Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies often suffer from a lack of signal-to-noise, requirements for extensive optimization for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein-protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting “activity-responsive RNAP” (AR) system to create light and small molecule activated biosensors, demonstrating the “plug-and-play” nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein-protein interactions and trigger RNA nanostructure production, protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biology applications.

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.

Analysis of naphthyridinomycin gene cluster revealed that this antibiotic is generated by nonribosomal peptide synthetase (NRPS) machinery. However, four modules encoded by two genes do not correspond with the structural units in the final product. Genetic and biochemical characterization of the gene cluster suggested that the leader peptide mechanism for the NRPS assembly line was involved in biosynthesis of this tetrahydroisoquinoline alkaloid.