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

Base editors, e.g.,\ncytosine deaminases, are powerful tools for\nprecise DNA editing <i>in vivo</i>, enabling both targeted\nnucleotide conversions and segment-specific diversification of bacterial\ngenomes. Yet, regulation of their spatiotemporal activity is crucial\nto avoid off-target effects and enabling controlled evolution of specific\ngenes and pathways. This work reports a strategy for tight control\nof base-editing devices through subjecting their expression to a genetic\nAND logic gate in which two chemical inducer inputs are strictly required\nfor cognate activity. The case study involves an archetypal genetic\ndevice consisting of a cytosine deaminase (pmCDA1) fused to a T7 RNA\npolymerase (RNAP<sup>T7</sup>), which cause intensive diversification\nof DNA portions bordered by a T7 promoter and a T7 terminatorbut\nwhose activity <i>in vivo</i> has been shown unattainable\nto govern with standard conditional expression systems. By encoding\nup to three UAG stop codons into the DNA sequence of the pmCDA1-RNAP<sup>T7</sup> fusion, which is transcribed by the 3-methylbenzoate inducible\npromoter <i>Pm</i>, we first broke the structure of the\nhybrid protein. Then, to overcome the interruptions caused by UAG\ncodons, we placed transcription of a <i>supF</i> tRNA under\nthe control of a cyclohexanone-dependent system. When tested in the\nsoil bacterium and metabolic engineering chassis Pseudomonas\nputida KT2440, these modifications changed the performance\nof the sliding base editor from a flawed YES logic to a precise AND\nlogic. We also showed that such a 2-layer control brings about a minimal\nbackground activity as compared to a single-input digitalizer circuit.\nThese results show the ability of suppressor tRNA-based logic gates\nfor achieving stringent expression of otherwise difficult to control\ndevices.