AND Logic Based on Suppressor tRNAs Enables Stringent Control of Sliding Base Editors in Pseudomonas putida

Base editors, e.g., cytosine deaminases, are powerful tools for precise DNA editing in vivo, enabling both targeted nucleotide conversions and segment-specific diversification of bacterial genomes. Yet, regulation of their spatiotemporal activity is crucial to avoid off-target effects and enabling controlled evolution of specific genes and pathways. This work reports a strategy for tight control of base-editing devices through subjecting their expression to a genetic AND logic gate in which two chemical inducer inputs are strictly required for cognate activity. The case study involves an archetypal genetic device consisting of a cytosine deaminase (pmCDA1) fused to a T7 RNA polymerase (RNAP^T7), which cause intensive diversification of DNA portions bordered by a T7 promoter and a T7 terminator─but whose activity in vivo has been shown unattainable to govern with standard conditional expression systems. By encoding up to three UAG stop codons into the DNA sequence of the pmCDA1-RNAP^T7 fusion, which is transcribed by the 3-methylbenzoate inducible promoter Pm, we first broke the structure of the hybrid protein. Then, to overcome the interruptions caused by UAG codons, we placed transcription of a supF tRNA under the control of a cyclohexanone-dependent system. When tested in the soil bacterium and metabolic engineering chassis Pseudomonas putida KT2440, these modifications changed the performance of the sliding base editor from a flawed YES logic to a precise AND logic. We also showed that such a 2-layer control brings about a minimal background activity as compared to a single-input digitalizer circuit. These results show the ability of suppressor tRNA-based logic gates for achieving stringent expression of otherwise difficult to control devices.