Florian Hoffmann, Ph.D.

All Publications

• Structural basis of RNA-guided transcription by a dCas12f-σE-RNAP complex. bioRxiv : the preprint server for biology Xiao, R., Hoffmann, F. T., Xie, D., Wiegand, T., Palmieri, A. I., Sternberg, S. H., Chang, L. 2025

  Abstract

  RNA-guided proteins have emerged as critical transcriptional regulators in both natural and engineered biological systems by modulating RNA polymerase (RNAP) and its associated factors1-5. In bacteria, diverse clades of repurposed TnpB and CRISPR-associated proteins repress gene expression by blocking transcription initiation or elongation, enabling non-canonical modes of regulatory control and adaptive immunity1,6,7. Intriguingly, a distinct class of nuclease-dead Cas12f homologs (dCas12f) instead activates gene expression through its association with unique extracytoplasmic function sigma factors (σE)8, though the molecular basis has remained elusive. Here we reveal a novel mode of RNA-guided transcription initiation by determining cryo-electron microscopy structures of the dCas12f-σE system from Flagellimonas taeanensis. We captured multiple conformational and compositional states, including the DNA-bound dCas12f-σE-RNAP holoenzyme complex, revealing how RNA-guided DNA binding leads to σE-RNAP recruitment and nascent mRNA synthesis at a precisely defined distance downstream of the R-loop. Rather than following the classical paradigm of σE-dependent promoter recognition, these studies show that recognition of the -35 element is largely supplanted by CRISPR-Cas targeting, while the melted -10 element is stabilized through unusual stacking interactions rather than insertion into the typical recognition pocket. Collectively, this work provides high-resolution insights into an unexpected mechanism of RNA-guided transcription, expanding our understanding of bacterial gene regulation and opening new avenues for programmable transcriptional control.

  View details for DOI 10.1101/2025.06.10.658880

  View details for PubMedID 40661421

  View details for PubMedCentralID PMC12259091

• Exapted CRISPR-Cas12f homologs drive RNA-guided transcription. bioRxiv : the preprint server for biology Hoffmann, F. T., Wiegand, T., Palmieri, A. I., Glass-Klaiber, J., Xiao, R., Tang, S., Le, H., Meers, C., Lampe, G. D., Chang, L., Sternberg, S. H. 2025

  Abstract

  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 (σE) 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 σE factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically-linked dCas12f and σE 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 σE was dependent on dCas12f and guide RNA (gRNA), suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σE ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σE 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 humans4,5.

  View details for DOI 10.1101/2025.06.10.658865

  View details for PubMedID 40661409

  View details for PubMedCentralID PMC12259090

• Temperate phages enhance host fitness via RNA-guided flagellar remodeling. bioRxiv : the preprint server for biology Walker, M. W., Richard, E., Wiegand, T., Wang, J., Yang, Z., Casas-Ciniglio, A. A., Hoffmann, F. T., Shahnawaz, H., Gaudet, R. G., Arpaia, N., Fernández, I. S., Sternberg, S. H. 2025

  Abstract

  Bacterial flagella drive motility and chemotaxis while also playing critical roles in host-pathogen interactions, as their oligomeric subunit, flagellin, is specifically recognized by the mammalian immune system and flagellotropic bacteriophages. We recently discovered a family of phage-encoded, RNA-guided transcription factors known as TldR that regulate flagellin expression. However, the biological significance for this regulation, particularly in the context of host fitness, remained unknown. By focusing on a human clinical Enterobacter isolate that encodes a Flagellin Remodeling prophage (FRφ), here we show that FRφ exploits the combined action of TldR and its flagellin isoform to dramatically alter the flagellar composition and phenotypic properties of its host. This transformation has striking biological consequences, enhancing bacterial motility and mammalian immune evasion, and structural studies by cryo-EM of host- and prophage-encoded filaments reveal distinct architectures underlying these physiological changes. Moreover, we find that FRφ improves colonization in the murine gut, illustrating the beneficial effect of prophage-mediated flagellar remodeling in a host-associated environment. Remarkably, flagellin-regulating TldR homologs emerged multiple times independently, further highlighting the strong selective pressures that drove evolution of RNA-guided flagellin control. Collectively, our results reveal how RNA-guided transcription factors emerged in a parallel evolutionary path to CRISPR-Cas and were co-opted by phages to remodel the flagellar apparatus and enhance host fitness.

  View details for DOI 10.1101/2025.07.22.666180

  View details for PubMedID 40777476

  View details for PubMedCentralID PMC12330518

• TnpB homologues exapted from transposons are RNA-guided transcription factors. Nature Wiegand, T., Hoffmann, F. T., Walker, M. W., Tang, S., Richard, E., Le, H. C., Meers, C., Sternberg, S. H. 2024; 631 (8020): 439-448

  Abstract

  Transposon-encoded tnpB and iscB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination1-4. These widespread gene families were repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12 (refs. 5,6). We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR-Cas adaptive immunity. Here, using phylogenetics, structural predictions, comparative genomics and functional assays, we uncover multiple independent genesis events of programmable transcription factors, which we name TnpB-like nuclease-dead repressors (TldRs). These proteins use naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPR interference technologies invented by humans7. Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility8, phage susceptibility9, and host immunity10. Collectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of transposon-encoded genes, and reveals the evolutionary trajectory of diverse RNA-guided transcription factors.

  View details for DOI 10.1038/s41586-024-07598-4

  View details for PubMedID 38926585

  View details for PubMedCentralID PMC11702177

• Transposon-encoded nucleases use guide RNAs to promote their selfish spread. Nature Meers, C., Le, H. C., Pesari, S. R., Hoffmann, F. T., Walker, M. W., Gezelle, J., Tang, S., Sternberg, S. H. 2023; 622 (7984): 863-871

  Abstract

  Insertion sequences are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance1. IS200- and IS605-family transposons undergo 'peel-and-paste' transposition catalysed by a TnpA transposase2, but they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively3,4. Recent studies have demonstrated that TnpB and IscB function as RNA-guided DNA endonucleases5,6, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB and IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related insertion sequences from Geobacillus stearothermophilus that encode several TnpB and IscB orthologues, and showed that a single TnpA transposase was broadly active for transposon mobilization. The donor joints formed upon religation of transposon-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB and IscB nucleases, and co-expression of TnpB and TnpA led to substantially greater transposon retention relative to conditions in which TnpA was expressed alone. Notably, TnpA and TnpB also stimulated recombination frequencies, surpassing rates observed with TnpB alone. Collectively, this study reveals that RNA-guided DNA cleavage arose as a primal biochemical activity to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defence.

  View details for DOI 10.1038/s41586-023-06597-1

  View details for PubMedID 37758954

  View details for PubMedCentralID PMC11758364

• Selective TnsC recruitment enhances the fidelity of RNA-guided transposition. Nature Hoffmann, F. T., Kim, M., Beh, L. Y., Wang, J., Vo, P. L., Gelsinger, D. R., George, J. T., Acree, C., Mohabir, J. T., Fernández, I. S., Sternberg, S. H. 2022; 609 (7926): 384-393

  Abstract

  Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD1, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs2,3. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins4. How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous-hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.

  View details for DOI 10.1038/s41586-022-05059-4

  View details for PubMedID 36002573

  View details for PubMedCentralID PMC10583602