Exapted CRISPR-Cas12f homologs drive RNA-guided transcription

Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP)¹. Although the seven σ factors in E. coli have been extensively characterized², Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ^E) whose precise roles are unknown, pointing to additional layers of regulatory potential³. Here we uncover an unprecedented mechanism of RNA-guided gene activation involving the coordinated action of σ^E factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically-linked dCas12f and σ^E homologs in E. coli using RIP-seq and ChIP-seq experiments, revealing systems that exhibited robust guide RNA enrichment and DNA target binding with a minimal 5'-G target-adjacent motif (TAM). Recruitment of σ^E was dependent on dCas12f and guide RNA (gRNA), suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σ^E ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σ^E complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription (RGT) that embodies natural features reminiscent of CRISPRa technology developed by humans^4,5.