Extended Data Fig. 10: Mechanisms of bacterial gene activation and repression, including newly discovered, RNA-guided pathways driven by dCas12f and TldR.

RNA-based mechanisms (top row) can activate gene expression, such as the RprA small RNA (sRNA) activating rpoS by relieving secondary structure inhibition, or repress gene expression, such as the RyhB sRNA destabilizing sodB through base pairing and RNase recruitment. Protein-based mechanisms (second row from top) can activate gene expression, such as Crp (cAMP receptor protein) enhancing RNAP recruitment at the envZ promoter, or repress gene expression, such as HTH transcription factors binding DNA to block RNAP-promoter recognition. σ factor-based mechanisms (second row from bottom) can activate gene expression, such as extracytoplasmic function (ECF) σ^E factors recruiting RNAP to specific promoters to drive transcription, or repress gene expression, such as when their activities are inhibited by FecR anti-σ factors. Finally, we report novel RNA-guided pathways of gene regulation (bottom row). In previous work, we uncovered TnpB-like nuclease-dead repressors (TldR) that exploit gRNAs to bind complementary DNA target sites, thereby preventing promoter recognition by RNAP (bottom right). In this study, we uncover nuclease-dead Cas12f proteins that exploit gRNAs to bind complementary DNA target sites and directly recruit σ^E factors and RNAP, thereby driving promoter-independent transcription of diverse genetic operons such as susCD polysaccharide utilization loci (bottom left). Collectively, our work highlights a new axis of gene regulation control via exapted, RNA-guided transcription factors akin to CRISPRi and CRISPRa.