Autocatalytic base editing for RNA-responsive translational control.

Genetic circuits that control transgene expression in response to pre-defined transcriptional cues would enable the development of smart therapeutics. To this end, here we engineer programmable single-transcript RNA sensors in which adenosine deaminases acting on RNA (ADARs) autocatalytically convert target hybridization into a translational output. Dubbed DART VADAR (Detection and Amplification of RNA Triggers via ADAR), our system amplifies the signal from editing by endogenous ADAR through a positive feedback loop. Amplification is mediated by the expression of a hyperactive, minimal ADAR variant and its recruitment to the edit site via an orthogonal RNA targeting mechanism. This topology confers high dynamic range, low background, minimal off-target effects, and a small genetic footprint. We leverage DART VADAR to detect single nucleotide polymorphisms and modulate translation in response to endogenous transcript levels in mammalian cells.

Redesign of ultrasensitive and robust RecA gene circuit to sense DNA damage.

SOS box of the recA promoter, PVRecA from Vibrio natriegens was characterized, cloned and expressed in a probiotic strain E. coli Nissle 1917. This promoter was then rationally engineered according to predicted interactions between LexA repressor and PVRecA . The redesigned PVRecA-AT promoter showed a sensitive and robust response to DNA damage induced by UV and genotoxic compounds. Rational design of PVRecA coupled to an amplification gene circuit increased circuit output amplitude 4.3-fold in response to a DNA damaging compound mitomycin C. A TetR-based negative feedback loop was added to the PVRecA-AT amplifier to achieve a robust SOS system, resistant to environmental fluctuations in parameters including pH, temperature, oxygen and nutrient conditions. We found that E. coli Nissle 1917 with optimized PVRecA-AT adapted to UV exposure and increased SOS response 128-fold over 40 h cultivation in turbidostat mini-reactor. We also showed the potential of this PVRecA-AT system as an optogenetic actuator, which can be controlled spatially through UV radiation. We demonstrated that the optimized SOS responding gene circuits were able to detect carcinogenic biomarker molecules with clinically relevant concentrations. The ultrasensitive SOS gene circuits in probiotic E. coli Nissle 1917 would be potentially useful for bacterial diagnosis.