Genetic Biosensor for Optimizing Double-Stranded RNA Production by Bacteria

Bacteria can be engineered to produce double-stranded RNA (dsRNA) molecules that induce a targeted RNA interference (RNAi) response in plants and animals for applications ranging from pest control to functional genomics. We developed a genetically encoded sensor that uses bimolecular fluorescence complementation to report relative dsRNA levels within bacterial cells. We tested sensor designs consisting of fusions of different dsRNA-binding domains derived from viruses to fragments of a split fluorescent protein in Escherichia coli. Then, we used the optimized dsRNA sensor design to demonstrate enhanced dsRNA accumulation in engineered strains of the aphid symbiont Serratia symbiotica, including a new RNase III deletion mutant. Our biosensor provides a convenient fluorescent readout that can be used to accelerate the design-build-test cycle for maximizing dsRNA yields in bacteria, including species native to plant and animal microbiomes that can be used to implement symbiont-mediated RNAi.