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). Although the seven σ factors in Escherichia coli have been extensively characterized, Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ) whose precise roles are unknown, pointing to additional layers of regulatory potential. Here we uncover a mechanism of RNA-guided gene activation involving the coordinated action of σfactor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically linked dCas12f and σhomologues in E. coli using RNA and chromatin immunoprecipitation experiments, revealing systems that exhibit robust guide RNA enrichment and DNA target binding with a minimal 5'-G target-adjacent motif. Recruitment of σwas dependent on dCas12f and guide RNA, suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σcomplexes 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 that embodies natural features reminiscent of CRISPR activation (CRISPRa) technology. 1 2E3E E E E E4,5
CRISPR Gene Editing