Sternberg Lab

16/16 This work was a big team effort and fun collaboration! Thanks to everyone: Matt, Egill, Tanner, Jing, Zaofeng, Americo, Florian, Hamna, Ryan, Nick, Israel and our stellar mentor Sam for making this project possible!

15/16 In summary, we shed light on the biological role of a naturally occurring version of CRISPR interference (CRISPRi), that emerged in gut-associated bacteria to benefit multiple aspects of their lifestyle.

14/16 The repeated exaptation of TnpB to regulate flagellin suggests strong evolutionary pressure favoring RNA-guided control of flagellin expression, particularly in gut-associated bacterial species.

13/16 In other key members of the human gut microbiome, flagellin-regulating TldRs can be found additionally associated with the translational repressor CsrA. The coordinated action of TldR and CsrA suggests a dual mode of both transcriptional and post-transcriptional gene regulation.

12/16 The extra domain stabilizes the filament by increasing inter-subunit contacts. Structure-guided mutagenesis confirmed that this domain is essential to improve motility of the lysogen. We believe it could similarly explain the decreased activation of TLR5.

11/16 We found that both flagellar filaments have a drastically different structure. The prophage flagellar filament is significantly thicker and presents a surprising mesh-like structure. This is due to the presence of an extra domain in the prophage flagellin.

10/16 To try to link these phenotypic observations, we teamed up once again with @piratefernandez.bsky.social‬ and decided to inspect the structures of the prophage flagellin in comparison to their host counterpart.

9/16 The phenotypic effects of lysogenization result in a consequential ecological outcome: enhanced colonization of the murine gut, illustrating the advantage of flagellar remodeling in a host-associated environment.

8/16 We also find that lysogenic conversion by FRφ reduces activation of TLR5, a receptor that detects flagellin to initiate the immune response, suggesting that lysogenization may facilitate immune evasion.

7/16 We find that lysogenic conversion by FRφ leads to increased motility of the host. Essentially, Enterobacter (and even E. coli actually) swims faster when expressing FliCp instead of FliCh.

6/16 To tackle this question we focused on a clinical isolate of Enterobacter, that natively contains what we now call a Flagellin Remodeling prophage (FRφ), and started our detective work to uncover the potential advantage(s) conferred by the acquisition of this prophage.

5/16 Prophages sometimes provide new traits to their bacterial host, that is called “lysogenic conversion”. Now, why would a phage manipulate the flagellar apparatus of their host? That question is the focus of our new study.

4/16 Strikingly, FliC-associated TldRs are found in prophages and target the promoter of host-encoded FliC. Basically, TldR silences the host FliC (FliCh) and the prophage copy (FliCp) takes over, effectively leading to a remodeling of the host flagellar apparatus.

3/16 Unlike TnpB, which can be found in transposons and in association with transposases, TldR is often found in association with FliC (flagellin), the main subunit of flagellar filaments. Flagella are large appendages used by bacteria for motility.

2/16 Last year we reported the discovery of RNA-guided transcriptional repressors, derived from the Cas12 ancestor TnpB. We called it TldR for “TnpB-like nuclease dead repressors”. Yet, we did not address their actual biological role.
More details here: tinyurl.com/2camf88e

1/16 New pre-print from the Sternberg Lab!
We uncover how temperate phages can use RNA-guided transcription factors to remodel the flagellar composition of their bacterial host and enhance their fitness.
Find the preprint and full story here: tinyurl.com/mshwjd77

Q&A: One scientist’s bold vision to make on-demand treatments routine for life-threatening rare genetic diseases Broad core member and gene editing pioneer David Liu describes a framework that could enable the treatment of 1,000 patients with personalized gene editing therapies by 2030.

Gene editing pioneer David Liu discusses baby K.J. Muldoon, the first person treated with a customized gene editing therapy, what made K.J.'s treatment possible, and how on-demand treatments for rare genetic diseases could one day become routine. #GeneEditing #Science

Barbara McClintock portrait

🧬🌽 Happy Transposon Day! 🌽🧬

Today we celebrate the birthday of Barbara McClintock - scientist extraordinaire and discoverer of jumping genes. Still the only woman to have an unshared Nobel Prize in the biomedical sciences #TransposonDay2025

Nature invented CRISPRa way before scientists did 🤩

Awesome work by the @sternberglab.bsky.social

Looking forward to seeing cool applications coming out of this !

Did you think you needed a promoter for CRISPRa? Think again!

10/10 This work was a big team effort and fun collaboration! Thanks to everyone: Florian, Tanner, Adriana, Juniper, Renjian, ‪@stephentang23.bsky.social‬, Henry, Chance, George, and our stellar mentors Sam and Leifu for making these projects possible!

9/10 In summary, we discovered an RNA-guided system that programmably creates de novo transcription start sites in bacteria. It represents a naturally occurring version of CRISPR activation (CRISPRa) but does not rely on targeting pre-existing promoters.

Structural basis of RNA-guided transcription by a dCas12f-σE-RNAP complex RNA-guided proteins have emerged as critical transcriptional regulators in both natural and engineered biological systems by modulating RNA polymerase (RNAP) and its associated factors(1-5). In bacter...

8/10 We teamed up with the Chang Lab who determined the beautiful structures of the RNA-guided transcription initiation complex (and more!) which feature dCas12f, Sigma and the native RNA polymerase. Be sure to check out their thread on the structural findings! www.biorxiv.org/content/10.1...

7/10 Transcription initiates in a tight ~45-48 bp window downstream of the PAM/TAM flanking the target site. All sequence specificity is imparted by Cas12f. The associated Sigma factor is recruited without imposing any of the typical -35 and -10 promoter DNA sequence requirements.

6/10 dCas12f-Sigma factors are able to recruit their native RNA polymerase (RNAP) to drive RNA-guided transcription at programmable sites. Remarkably, we were able to generate RNA (see RNA-seq) without relying on promoters in intergenic sequences, even within genes, and on either DNA strand.

5/10 Indeed, when Cas12f is programmed to target various sites in the E. coli genome, we also observe recruitment of the Sigma factor to the same sites. dCas12f and Sigma appear to form a co-complex! Spoiler: check out our companion structure article with the Chang Lab!

4/10 Sigma factors are essential for driving bacterial transcription by RNA polymerase (RNAP) from specific promoter sequence motifs. dCas12f-associated Sigma factors have an atypical, extended C-terminal domain, potentially facilitating interaction between Sigma and Cas12f.

3/10 The dead-Cas12f is highly compact (~370 aa) and binds a natural single-guide RNA (~90 nt). Strikingly, dCas12f has minimal PAM/TAM requirements. It can target custom sites flanked by a simple 5’-G nucleotide.

2/10 Cas proteins typically defend against bacterial viruses by cleaving their genomes. The Cas12f homologs we identified in bacteria (see also Altae-Tran et al. 2023) are nuclease-dead (dCas12f) and have a conserved association with SigmaE factors (RpoE), suggesting a new function.

1/10 New pre-print(s) from the Sternberg Lab in collaboration with Leifu Chang's Lab! We uncover the unprecedented molecular mechanism of CRISPR-Cas12f-like proteins, which drive RNA-guided transcription independently of canonical promoter motifs.
Full story here:
www.biorxiv.org/content/10.1...