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

Base editors, e.g., cytosine deaminases, are powerful tools for precise DNA editing <i>in vivo</i>, 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 <i>in vivo</i> 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 <i>Pm</i>, we first broke the structure of the hybrid protein. Then, to overcome the interruptions caused by UAG codons, we placed transcription of a <i>supF</i> tRNA under the control of a cyclohexanone-dependent system. When tested in the soil bacterium and metabolic engineering chassis <i>Pseudomonas putida</i> 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.