Antibiotic resistance through horizontal gene transfer is a major challenge in microbiology and medicine. We describe a CRISPR-based screening system that couples spacer adaptation with gene repression to identify bacterial genes that sensitize bacteria to antibiotics. We used this screening system in Escherichia coli, on seven different antibiotics to select spacers that conferred resistance to them. The screen identified both known and previously unrecognized genes that modulate antibiotic susceptibility. We defined this collection of genes that sensitize bacteria to antibiotics as the 'sensitasome'. Among the identified targets were the essential genes gyrA and gyrB, whose repression increased resistance to multiple antibiotics. Some sensitization genes exhibited a clear trade-off: their repression conferred resistance to certain antibiotics while increasing susceptibility to others. Sensitasome-targeting spacers were also effective in Shigella and Salmonella, indicating that the resistance mechanism is conserved across species. Finally, we repurposed a selected spacer as a CRISPR-based antibiotic selection marker using the compact Cas12m effector. Together, these findings establish adaptation-coupled CRISPRi as a simple platform for discovering antibiotic sensitization genes and provide an environmentally safer alternative to conventional antibiotic resistance markers.

In Streptococcus pneumoniae, the RpiR transcriptional regulator NanR (SpNanR) senses sialic acid in the environment and upregulates transcription of the nan and sia A operons to increase uptake and metabolism of sialic acid. The molecular basis of this activation is unknown. Here, we demonstrate that SpNanR binds N-acetylmannosamine-6-phosphate, a metabolite of sialic acid catabolism. SpNanR exists in a dimer-tetramer equilibrium, and N-acetylmannosamine-6-phosphate binding strongly stabilizes the tetramer. Crystal structures and site-specific substitutions demonstrate that N-acetylmannosamine-6-phosphate bridges and stabilizes the SpNanR tetramer. SpNanR binds its DNA recognition sequence with nanomolar affinity. Notably, the effector N-acetylmannosamine-6-phosphate does not affect the affinity of SpNanR for DNA. The DNA-binding domains are not structurally coupled to the sugar isomerase domains, explaining why N-acetylmannosamine-6-phosphate binding does not affect DNA binding. Structural analysis reveals that sequence specificity arises through distortion of B-DNA and an unusual π-stack formed by two arginine residues in the minor groove, while affinity is driven by backbone contacts. We propose a mechanism by which S. pneumoniae regulates sialic acid metabolism, consistent with our biophysical experiments and in vivo regulatory behavior. These findings define a unique activation mechanism for an RpiR regulator and provide new insights into carbohydrate-responsive gene regulation in pneumococci.

Single-strand DNA breaks are among the most frequent forms of DNA damage, yet how PARP2 recognizes these lesions within the constrained geometry of chromatin remains unclear. Here we use cryogenic electron microscopy (cryo-EM) to determine structures of PARP2 and HPF1 bound to nucleosomes containing a site-specific single-strand break (SSB) at superhelical location + 5.3. The structures reveal a multivalent binding mode in which the Trp-Gly-Arg (WGR) domain engages the break while the helical domain and HPF1 contact the opposing DNA gyre, spanning both DNA gyres of the nucleosome. This configuration explains the higher affinity of PARP2 for nucleosomal SSBs relative to free DNA. 3D classification resolves multiple PARP2-bound states with distinct WGR orientations and DNA backbone distortions, revealing structural plasticity during damage recognition. Comparative analysis shows that HPF1 constrains the conformational ensemble of PARP2 and promotes a defined catalytic domain architecture compatible with histone PARylation. PARP inhibitors (olaparib, talazoparib, EB-47) stabilize WGR-helical domain interfaces and make the ADP-ribosyltransferase-HPF1 catalytic module dynamic, consistent with reverse allosteric retention on damaged chromatin, while also promoting asymmetric nucleosomal DNA unwrapping. Together, these structures provide a chromatin-context framework for understanding PARP2 recognition of nucleosomal DNA breaks and how PARPis reshape these interactions to promote PARP retention and local chromatin remodeling.

Enhancer RNAs (eRNAs) contribute to gene expression specificity, yet their expression patterns at single-cell resolution remain poorly understood. Across 184 329 high-quality single cells from 187 full-length single-cell RNA-seq datasets, we identified 85 977 human and 82 727 mouse eRNAs, which were sparsely detected at the single-cell level (<5% in humans and <8% in mice) but showed high intercellular heterogeneity and contributed substantially to lineage-specific marker repertoires, accounting for up to 33.5% of human and 46.5% of mouse lineage markers. Through systematic analysis of eRNA expression patterns in embryonic development, brain tissues, and cancer, cell-specific eRNAs in different cell types were identified and corresponding regulatory networks were constructed. Our results indicate that eRNAs play important roles in diverse biological processes through modulation of gene expression. Finally, RACE confirmed the full transcription of a colorectal cancer-associated eRNA, whose elevated plasma levels, diagnostic performance, and postoperative reduction support its potential as a noninvasive biomarker for colon cancer detection and monitoring. Collectively, this study provides a comprehensive single-cell landscape of eRNA expression and offers a valuable resource for understanding eRNA-mediated regulatory mechanisms.

Proper segregation of meiotic chromosomes depends on the controlled formation and resolution of recombination intermediates that give rise to crossovers (COs). While the BTRR complex-comprising the BLM helicase, topoisomerase IIIα, and the proteins RMI1 and RMI2-is a well-characterized regulator of recombination in somatic cells, its function during meiosis remains incompletely understood, particularly for RMI2 in mammals. Here, we investigate the role of RMI2 in mouse spermatogenesis using a targeted RMI2 knockout model. Loss of RMI2 results in severe meiotic defects, including reduced testis size, increased apoptosis at the metaphase-anaphase I transition, and frequent chromosomal abnormalities such as lagging chromosomes in metaphase I cells and anaphase I chromatin bridges. Cytological analyses reveal disruptions in the processing of recombination intermediates. Our work monitoring molecular recombination outcomes at known recombination hotspots suggests that RMI2 regulates the balance of recombination pathways, constrains aberrant branch migration, and ensures proper CO maturation. Our findings show previously unappreciated functions for RMI2 essential for coordinating recombination intermediate metabolism and promoting proper chromosome conformation and segregation during meiosis. These results provide insights into the complex regulation of recombination intermediate homeostasis in higher eukaryotes and highlight the role of RMI2 in maintaining genome stability and proper gamete development in mammals.

CRISPR-Cas12a nucleases typically induce double-strand breaks (DSBs) to initiate genome editing. However, reliance on DSBs risks chromosomal rearrangements and unintended insertions or deletions at off-target sites. Therefore, engineering nickase variants that cleave only a single DNA strand might enable precise editing platforms. We describe an unbiased in vivo screen in Escherichia coli to identify LbCas12a nickase variants. We targeted the RuvC Lid domain for mutagenesis, hypothesizing that altering this region would disrupt the coordination of strand cleavage. While the screen produced many nickase variants with targeted point substitutions (Lid1), it also serendipitously yielded a variant (Lid2) with an unprecedented Lid domain in terms of size and sequence. Biochemical characterization confirms that Lid2 robustly nicks the non-target strand (NTS) with minor cleavage activity observed for the target strand (TS), accumulating predominantly nicked substrates. Cryo-electron microscopy (cryo-EM) at 2.5 Å resolution reveals that, in Lid2, the native α-helical Lid is replaced, uncoupling the concerted cleavage mechanism. Specifically, the remodeled Lid appears to stabilize the NTS in the active site, whereas it hampers the conformational transitions required for subsequent TS cleavage. This robust nickase-phenotype of this Cas12a variant expands the CRISPR-Cas toolbox and offers a new component for precise genome editing applications.

In mammalian cells, the addition of ADP-ribose to proteins and DNA plays well established roles in regulating cell function. Recently, RNA ribosylation was also found in mammalian cells under conditions of cell stress, though the functional consequences remain unclear. Here we find that infection with chikungunya virus, a positive strand RNA virus that causes frequent widespread epidemics, increases overall levels of RNA ribosylation in human fibroblasts. During infection, viral RNA is ribosylated by the PARP12 ribosyltransferase, which is counteracted by the viral nsP3 ribosylhydrolase. Increased viral RNA ribosylation resulted in decreased translation in cell-free systems and infected fibroblasts, and more rapid viral RNA decay. Further, ribosylated RNA potently induced the expression of antiviral host response genes. Together these data show the first functional consequences of RNA ribosylation in mammalian cells by showing that RNA ribosylation inhibits translation, decreases RNA stability, and creates a novel pathogen-associated molecular pattern that activates the host innate immune response. As macrodomains are present in multiple unrelated viruses, our data suggest RNA ribosylation is a novel component of cellular antiviral sensing pathway. These results define a framework for understanding how RNA ribosylation regulates RNA metabolism during viral infection.

Broad-host-range IncA and IncC (A/C) conjugative plasmids propagate multidrug resistance in bacteria and mobilize chromosomal resistance islands, including Salmonella Genomic Island 1 (SGI1), across genera. A/C plasmids mobilize SGI1 at very high frequencies and are inhibited by SGI1. Here, we show that SGI1 and kin encode a fertility inhibitor, which we named CtiC (for 'Conjugative Transfer Inhibition of IncC'), that hampers A/C plasmid transfer. Fertility inhibition facilitates the propagation of SGI1, which is known to be destabilized by coresident A/C plasmids. ctiC expression is both constitutive and activated during conjugative transfer of A/C plasmids. Structural predictions suggest that CtiC resembles the C-terminus of the MOBH12-family relaxase TraI of A/C plasmids. Bacterial two-hybrid assays and relaxase domain substitutions show that CtiC and the C-terminus of TraI interact with the plasmid-encoded mobilization factor MobI, which recognizes and binds the origin of transfer (oriT) of A/C plasmids. Hence, CtiC acts as a decoy that competes with TraI for binding to MobI, freeing TraI for efficient SGI1 mobilization, and precluding efficient processing of the plasmid's oriT. Our work reveals a mechanism of fertility inhibition that prevents relaxosome assembly and uncovers a functional domain in MOBH12 relaxases.

The Kaposi's sarcoma-associated herpesvirus oncoprotein vIRF3 is essential for primary effusion lymphoma (PEL) cell survival and cooperates with cellular interferon regulatory factor 4 (IRF4) to activate super-enhancers (SEs) controlling MYC and IRF4. However, the DNA sequence requirements for this cooperation are unknown. Using reporter assays, we mapped vIRF3/IRF4 responsiveness within the IRF4-SE to an ∼83-bp element activated by vIRF3 alone and cooperatively by vIRF3 and IRF4. vIRF3-dependent activation required an AP-1 motif, whereas cooperation with IRF4 required IRF4 DNA binding and IRF-related motifs outside canonical AP-1-IRF composite elements (AICEs). These motifs were necessary but insufficient outside their native sequence context, indicating a requirement for extended flanking sequences. DNA pulldowns revealed distinct motif requirements for vIRF3 and IRF4 association with the IRF4-SE. A distal MYC-SE required a similarly extended region containing a functional AICE2 motif, with vIRF3 activity depending primarily on its AP-1 half-site. Motif requirements were preserved in integrated reporter assays. BATF promoted vIRF3/IRF4-dependent activation preferentially in integrated reporters and was required to maintain vIRF3 and IRF4 occupancy at the endogenous MYC-SE. These findings define context-dependent DNA and cofactor requirements through which vIRF3 co-opts IRF4-regulated oncogenic enhancers in PEL.

PanGBank (https://pangbank.genoscope.cns.fr) is a comprehensive open-access database providing precomputed prokaryotic pangenomes at a broad taxonomic scale. Built upon PPanGGOLiN partitioned pangenome graphs, PanGBank addresses the growing need for large-scale comparative genomics through a standardized, regularly updated, and fully accessible resource. The initial release comprises two complementary collections covering >4600 prokaryotic species from the Genome Taxonomy Database (GTDB), encompassing over 393 000 genomes: GTDB_all, maximizing taxonomic and environmental diversity through the inclusion of metagenome-assembled genomes and single-cell amplified genomes, and GTDB_refseq, focusing on high-quality, annotation-rich genomes. Each species-level pangenome integrates graph-based statistical partitions into persistent, shell, and cloud gene families, together with regions of genomic plasticity (panRGP) and co-localized functional modules (panModule). PanGBank offers multiple access modes, including a REST API, a command-line interface, and an interactive web interface. By combining large-scale pangenome resources with advanced graph-based analyses, PanGBank provides a scalable framework for exploring microbial diversity, genome evolution, functional variation, and the dissemination of adaptive traits across prokaryotic populations, as illustrated by a use case on Acinetobacter baumannii pangenome investigating the distribution and evolution of antimicrobial resistance determinants.

We developed CUT&ID to simultaneously profile the native genomics and proteomics associations of any endogenous target, in a single workflow with no need for transgenesis. CUT&ID is enabled by a fusion protein of TurboID, protein A/G, and micrococcal nuclease, which drives sequential target recognition, proximal protein biotinylation, and DNA cleavage in living cells. CUT&ID is broadly applicable, works on fresh, frozen, or fixed nuclei, and provides high signal-to-noise genomics and proteomics identification across all types of gene regulators tested, including histone modifications, transcription factors, and non-DNA-binding proteins. CUT&ID's streamlined protocol yields a dual readout from 1 million cells per sample, making it an attractive alternative to existing methodologies.

Improving siRNA delivery to the central nervous system (CNS) is a major focus for treating the numerous debilitating neurological conditions that have a genetic basis. Here, we present an albumin-binding siRNA based on an amphiphilic dendrimer conjugate (D-siRNA). We demonstrate that D-siRNA achieves effective and homogeneous delivery throughout the CNS following administration into the cerebrospinal fluid (CSF). In mice, a single CSF administration of D-siRNA resulted in potent and durable gene silencing across various brain regions, with effects lasting 6 months without detectable toxicity. We validate its utility in larger rodents (rats) using intrathecal administration-a clinically relevant route-showing effective and broad delivery and robust silencing. Benchmarking against other clinically relevant siRNA delivery scaffolds revealed that D-siRNA provides comparable delivery and efficacy, with more efficient conversion of gross uptake to functional uptake. These findings support the use of albumin-binding conjugates for brain delivery, and position D-siRNA as a safe, effective, and durable platform for gene silencing in the CNS.

This study examines how DNA methylation by both phase-variable and canonical restriction-modification (RM) systems contributes to bacterial gene regulation, using the SpnD39III system of Streptococcus pneumoniae and the prototypical EcoKI system of Escherichia coli. RNA-seq analysis of pneumococcal phase-locked strains expressing individual SpnD39III specificities revealed widespread but promoter-context-dependent differential gene expression associated with methylation. Targeted luciferase reporter assays confirmed that methylation of specific intergenic sites can either activate or repress transcription, validating the transcriptomic observations. Genetic dissection demonstrated that these regulatory effects depend on methyltransferase activity mediated by HsdM and are independent of restriction endonuclease function. These methylation-dependent responses were conserved across pneumococcal strain backgrounds. Mechanistic analyses revealed that methylation alters DNA topology, with methylated plasmids exhibiting reduced negative supercoiling. Consistent with a topology-driven mechanism, modulation of the nucleoid-associated protein HlpA (HU) reshaped promoter responses in a manner dependent on methylation state and promoter topological responsiveness. Extension of these analyses to E. coli showed analogous methylation-dependent regulation by EcoKI, supported by reporter assays, quantitative polymerase chain reaction, RNA-seq, and methylome profiling. Together, these findings demonstrate that RM system-mediated DNA methylation functions as an epigenetic regulator across bacterial species by altering local DNA topology and promoter accessibility, thereby dynamically shaping bacterial gene regulatory networks.