Promoter-independent gene activation by Sigma factor-associated Cas12f proteins

Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (s) factors, leading to functional DNA engagement by RNA polymerase (RNAP). Although the 7 s factors in E. coli have been extensively characterized, Bacteroidetes species encode dozens of specialized s factors whose precise functions are unknown, suggesting additional layers of regulatory potential. Here we uncover an unprecedented mechanism of RNA-guided gene activation involving the coordinated action of sE factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically linked dCas12f and sE 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 surprisingly minimal 5'-G target-adjacent motif (TAM). Recruitment of sE was dependent on dCas12f and gRNA, suggesting a direct protein-protein complex, and co-expression experiments demonstrated that the ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-sE complexes drove potent gene expression in the absence of any requisite promoter motifs, with transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided gene activation that embodies natural features reminiscent of CRISPRa technology developed by humans. Overall design: E. coli ChIP-seq and RIP-seq profiling for 16 RNA-guided endonuclease-dead Cas12f proteins. ChIP-seq profiling for a subset of dCas12f-associated sE proteins. E. coli and F. taeanensis RNA-seq profiling.