Results 501-525 of about 1,000
  1. Bacteria often acquire resistance against antibiotics through the transfer of conjugative resistance plasmids. Hence, it is vital to develop strategies to mitigate the dispersal of antimicrobial resistance (AMR). CRISPR-based antimicrobial tools offer a sequence-specific solution to diminish and restrict the dissemination of antimicrobial resistance genes among bacteria. CRICON (CRISPR via conjugation) is an antimicrobial CRISPR tool that has been shown to efficiently reduce multi-resistance when targeting ESBL (Extended Spectrum Beta-Lactamase) harboring plasmids. However, conjugatively delivered genetic elements may be subjected to bacterial defense, lead to resistance development, and revert the efficiency of the CRISPR tools. Here, we studied the evolutionary consequences of four ESBL-harboring Escherichia coli strains targeted by CRICON in a 10-day multispecies microcosm experiment. We show that CRICON reduces the ESBL prevalence within the bacterial community, while the final ESBL persistence depends on the initial community composition. We observed an unexpected survival strategy of an ESBL-plasmid by escaping into a more competitive host species. Further, we show the development of partial resistance against the CRISPR-antimicrobials during the experiment. Our results underline the importance of the ecological and evolutionary factors in multispecies bacterial communities, as they may disrupt the effective use of CRISPR-based antimicrobial strategies via undesired outcomes of targeted therapies against plasmid-bearing multi-resistant bacteria.
    Date: 2025-04-15
    Authors: Given CJ, Jonsdottir I, Norvasuo K, Paananen P, Ruotsalainen P, Hiltunen T, Gunell M, Hakanen AJ, Jalasvuori M, Penttinen R.
    Ref: bioRxiv
  2. Type III CRISPR systems are defined by the presence of the Cas10 protein and are among the most abundant CRISPR systems in nature. Cas10 forms a complex with crRNA and several Cas proteins that surveils bacterial cells for foreign RNA molecules and when they are detected it activates a cascade of interference activities. The synthesis of the cyclic oligoadenylate signaling molecule by Cas10 is a key aspect of the interference cascade. Despite structures of the Cas10 complex bound to target RNAs, the molecular mechanism by which Cas10 senses the bound state to license interference is lacking. We identified five residues in S. epidermidis Cas10, two in the Cas10 Palm2 domain and three in domain 4, that line the target RNA binding channel. We assessed the contribution of these residues to interference in the context of a cognate or mismatched target RNA. We found that the residues regulate whether a mismatched crRNA-target RNA duplex is able to activate interference in vivo. We purified two site-directed mutants of Cas10-Csm and show with in vitro cOA synthesis assays they demonstrate enhanced discrimination of cognate versus mismatched targets.
    Date: 2025-04-15
    Authors: Khweis SA, Blackburn M, Pierce MO, Lewis CR, Dunkle JA.
    Ref: bioRxiv
  3. Clostridioides difficile is the major cause of nosocomial infections associated with antibiotic therapy. The severity of C. difficile infections increased worldwide with the emergence of hypervirulent strains, including 027 ribotype epidemic strains. Many aspects of C. difficile ’s adaptation strategies during pathogenesis remain poorly understood. This pathogen thrives in gut communities that are rich in microbes and phages. To regulate horizontal transfer of genetic material during its infection cycle, C. difficile relies on diverse mechanisms. More specifically, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas and Toxin-Antitoxin (TA) systems contribute to prophage maintenance, prevention of phage infection, and stress response. Abortive infection (Abi) systems can provide additional lines of anti-phage defense. RNAs have emerged as key components of these systems including CRISPR RNAs and antitoxin RNAs within type I and type III TA. We report here the identification of a new AbiF-like system within a prophage of the hypervirulent C. difficile strain R20291. It is associated with an Abi_2/AbiD/F protein family largely distributed in Bacillota and Pseudomonadota with structural links to ancestral Cas13 proteins at the origin of the RNA-targeting CRISPR-Cas13 systems. We demonstrated toxic activity of the AbiF Cd protein in C. difficile and in Escherichia coli and negative regulation of the abiF Cd expression by an associated non-coding RNA RCd22. RCd22 contains two conserved abiF motifs and is active both in cis and in trans to neutralize the toxin by direct RNA-protein interaction, similar to RNA antitoxin in type III TA. A mass spectrometry interactomics analysis of protein fractions from MS2-Affinity Purification coupled with RNA sequencing (MAPS) revealed the AbiF Cd protein among the most enriched RCd22 partners in C. difficile . Structural modeling of the RNA-protein complex and mutagenesis analysis revealed key positions on both protein and RNA partners for this interaction and toxic activity. In summary, these findings provide valuable insights into the mechanisms of interaction between bacteria and phages, which are pertinent to the advancement of phage therapy, genome editing, epidemiological surveillance, and the formulation of novel therapeutic approaches.
    Date: 2025-04-15
    Authors: Saunier M, Humbert A, Kreis V, Peltier J, Tisba A, Auxilien S, Blum M, Caldelari I, Lucier J, Ueda J, Gautheret D, Toffano-Nioche C, Andreani J, Fortier L, Soutourina O.
    Ref: bioRxiv
  4. Genetic screens are essential for uncovering novel molecular mechanisms and identifying the functions of hypothetical proteins. CRISPR interference (CRISPRi) is a powerful, programmable, and sequence-specific gene repression technology that can be used for high-throughput screening and targeted gene repression. Despite its ease of use, the initial development of CRISPRi systems is labor-intensive in many non-model organisms. Our goal is to simplify this by establishing a host-agnostic CRISPRi platform that utilizes the serine recombinase-assisted genome engineering (SAGE) system. This system integrates CRISPRi machinery directly into the bacterial chromosome, overcoming the limitations of plasmid-based systems and enabling wide sharing across diverse bacteria. We demonstrate the design and optimization of multiplexed CRISPRi to repress multiple genes simultaneously in phylogenetically distant bacteria. We use a Francisella novicida -derived Cas12a system that processes multiple distinct CRISPR RNAs, each targeting a unique gene sequence, from a single transcript. This allows easy multi-gene repression. By reinforcing gene repression with multiple guides targeting a single gene, we achieve robust genetic perturbations without the need to pre-screen the efficacy of guide RNAs. Using this toolkit, we perturb multiple combinations of growth and visual phenotypes in Pseudomonas fluorescens and demonstrate simultaneous repression of multiple fluorescent proteins to near background levels in bacteria from various other genera. While the tools are directly portable to all SAGE-compatible microbes, we illustrate the utility of SAGE by optimizing CRISPRi performance in Rhodococcus jostii through a combinatorial screen of Cas protein and CRISPR array expression variants. The efficient integration of CRISPRi machinery via the SAGE system paves the way for versatile genetic screening, enabling profound insights into gene functions both in laboratory conditions and relevant naturalistic scenarios.
    Date: 2025-04-15
    Authors: Elmore JR, Shrestha R, Wilson A, Fossen EV, Frank A, Francis RM, Baldino H, Stephenson M, Gupta B, Rivera J, Egbert RG.
    Ref: bioRxiv
  5. E3 ubiquitin ligases mediating turnover of proteins engaged in cancer progression point to key regulatory nodes. To uncover modifiers of metastatic competency, we conducted an in vivo genome-wide CRISPR-inactivation screen using cultured breast circulating tumor cells, following intravascular seeding and lung colonization. We identified HECTD4, a previously uncharacterized gene encoding a conserved potential HECT domain-containing ubiquitin transferase, as a potent tumor and metastasis suppressor. We show that purified HECTD4 mediates ubiquitin conjugation in vitro, and proteomic studies combined with ubiquitin remnant profiling identify a major degradation target as the prostaglandin synthetic enzyme cyclooxygenase-2 (COX-2; PTGS2). In addition to COX-2 itself, HECTD4 targets its regulatory kinase MKK7. In breast cancer models, HECTD4 expression is induced as cells lose adherence to the matrix, and its depletion massively increases COX-2 expression, enhancing anchorage-independent proliferation and tumorigenesis. Genetic or pharmacologic suppression of COX-2 reverses the pro-tumorigenic and pro-metastatic phenotype of HECTD4-depleted cells. Thus, HECTD4 encodes an E3 ubiquitin ligase that downregulates COX-2 suppressing anchorage-independence in epithelial cancer cells. Significance Statement A genome-wide CRISPR-inactivation screen identified the previously uncharacterized E3 ubiquitin ligase HECTD4, as a tumor and metastasis suppressor, with COX-2 as its major degradation target. The pro-tumorigenic and pro-metastatic effect of HECTD4 suppression depends on COX-2 stabilization, which is critical for anchorage-independent growth, providing a basis for investigating COX-2 inhibition to prevent metastatic recurrence.
    Date: 2025-04-15
    Authors: Vuille JA, Tanriover C, Micalizzi DS, Ebright RY, Animesh S, Morris R, Hajizadeh S, Nicholson ZJ, Russell HC, Zaniewski EF, Wittner BS, Wesley BK, Grünewald J, Szalay RN, Antmen E, Fox DB, Yang M, Joung JK, Gulhan DC, Elia AE, Haas W, Oh E, Maheswaran S, Haber DA.
    Ref: bioRxiv
  6. Abstract BAP1-deficient melanocytic tumors exhibit strong immunosuppressive features and poor prognosis. Currently, no immune-competent preclinical models exist to study their tumor-immune interactions or test new immunotherapies. This limitation hinders progress in understanding how BAP1 loss drives tumor aggressiveness and immune evasion. To address this, we generated a syngeneic BAP1 knockout melanocyte tumor line using CRISPR-Cas9. We then evaluated its functional and immunological impact in immune-competent mice, including its ability to recapitulate metabolic and immunosuppressive features of human BAP1-deficient melanomas. The selected knockout clone exhibited hallmarks of aggressive skin and intraocular melanomas, including epithelioid morphology, in vivo tumorigenic potential, rapid growth, and key immunosuppressive features, mirroring those observed in human BAP1-deficient melanomas. Cross-species single-cell transcriptome analysis demonstrated strong molecular overlap between BAP1 knockout mouse tumors and high-risk (class 2) human uveal melanomas, highlighting shared pathways in lipid metabolism, transmembrane receptor signaling, and immune modulation. Gene Set Enrichment Analysis confirmed that lipid metabolic reprogramming, previously described in human tumors, is also a key feature of our model, validating its ability to recapitulate human disease biology. This study introduces a syngeneic preclinical model that mimics the immunosuppressive landscape of BAP1-deficient melanocytic tumors, enabling the development and optimization of new combination immunotherapies.
    Date: 2025-04-15
    Authors: Wang MM, Li Y, Ho C, Yu W, Coupland S, Chan A, Figueiredo C.
    Ref: Research Square
  7. Long-read RNA sequencing has been broadly utilized to examine the diversity of transcriptomes, understand differential expression and discover novel transcript isoforms. One of the major limitations of whole transcriptome sequencing is the difficulty in obtaining sufficient depth for low abundant transcripts. Methods which address this are either difficult to scale or customize: long- range PCR is customizable but difficult to scale beyond a few targets; probe hybridization panels are suited for scaling but require substantial investment to customize. In this study, we adopted RNA-guided CRISPR-Cas9 nuclease-based enrichment to target specific human and SARS-CoV-2 transcripts followed by long-read sequencing, utilizing minimal number of guide RNAs per target isoform. Our findings demonstrate that the CRISPR-Cas system is a highly effective method for customizable long-read sequencing of target transcripts while maintaining the accuracy of relative gene expression levels. The results highlight a valuable method for future research on transcript enrichment for isoform identification and low abundance transcript detection in infectious disease diagnosis.
    Date: 2025-04-14
    Authors: Nguyen AN, Zhang J, Zhang S, Pitt ME, Ganesamoorthy D, Fritzlar S, Chang JJ, Londrigan SL, Coin LJ.
    Ref: bioRxiv
  8. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas technology has revolutionized molecular biology and therapeutic research. In this review, we highlight a focused analysis of CRISPR-Cas applications in the treatment and study of five major human diseases based on select peer-reviewed articles. While not exhaustive, our investigation provides insight into trends, mutation targets, and experimental outcomes, highlighting the evolving landscape of CRISPR as a therapeutic tool. We also discuss challenges, emerging tools like Cas12, Cas13, and Cas15, and future directions.
    Date: 2025-04-14
    Authors: Shree P.
    Ref: Preprints.org
  9. Genetic defects in AP2M1 , which encodes the μ-subunit of the adaptor protein complex 2 (AP-2) essential for clathrin-mediated endocytosis (CME), cause a rare form of developmental and epileptic encephalopathy (DEE). In this study, we modeled AP2M1 -DEE in Drosophila melanogaster to gain deeper insights into the underlying disease mechanisms. Pan-neuronal knock-down of the Drosophila AP2M1 ortholog, AP-2µ , resulted in a consistent heat-sensitive paralysis phenotype and altered morphology in class IV dendritic arborization (c4da) neurons. Unexpectedly, affected flies were resistant to antiseizure medications and exhibited increased resistance to electrically induced seizures. A CRISPR-engineered fly line carrying the recurrent human disease variant p.Arg170Trp displayed a milder seizure resistance phenotype. While these findings contrast with the human phenotype, they align with previous studies on other CME-related genes in Drosophila . Our results suggest that hyperexcitability and seizures in AP2M1 -DEE may stem from broader defects in neuronal development rather than direct synaptic dysfunction.
    Date: 2025-04-14
    Authors: Karge RA, Fischer FP, Schüth H, Wechner A, Peter S, Kilo L, Dichter M, Voigt A, Tavosanis G, van Loo KMJ, Koch H, Weber YG, Wolking S.
    Ref: bioRxiv
  10. Consumptive hypothyroidism, a rare pediatric disorder, arises from aberrant Dio3 expression, which inactivates thyroid hormones and disrupts skeletal development. This study investigates the regulatory role of the long non-coding RNA Dio3os , which activates Dio3 in cis and suppresses osteoblast differentiation in trans. We focused on mouse Dio3os variant 203 and human variants 203 and 205. Chromatin accessibility and histone modification analyses revealed higher chromatin openness and active histone H3 modifications at the Dio3os promoter and exons 2–3, followed by increased RUNX2 binding with reduced histone H3 modifications during maturation. Dio3os expression is enhanced by HDAC1/2, HIF1α, and thyroid hormones, but repressed by BMP2, TGFβ, Runx2, and Brg1. Overexpression of Dio3os upregulated Dio3 while downregulating osteogenic markers. CRISPR-mediated deletion of Dio3os exons or premature intronic polyadenylation suppressed Dio3 and restored osteogenic gene expression. RNA-seq and ATAC-seq confirmed enhanced thyroid hormone-responsive osteoblast gene activity in Dio3os -CRISPR-knockout cells. These findings reveal Dio3os as a key regulator of thyroid hormone metabolism and bone formation, presenting a novel target for treating skeletal abnormalities in early childhood hypothyroidism. Synopsis During hypothyroidism, long non-coding RNA (lncRNA) Dio3os activates neighboring Dio3 and inhibits non-neighboring OB-specific genes. In bone cells, the long non-coding RNA (lncRNA) Dio3os recruits the NuRD complex to the Dio3 gene promoter, activating its transcription through an unidentified deacetylation mechanism. Dio3os teams with NuRD, reverse T3 (rT3), and TRα1 bind together at the promoter’s thyroid-responsive element (TRE), replacing T3 to repress osteoblast gene transcription. These findings enhance our understanding of how hypothyroidism affects bone phenotypes.
    Date: 2025-04-14
    Authors: Chen Y, Wildman B, Rehan M, Mishra S, Hassan Q.
    Ref: bioRxiv
  11. The distribution of proteins across the plasma membrane is not uniform; however, the principles governing their organization remain not fully understood. Hypersensitive-induced reaction (HIR) proteins are plant-specific members of the stomatin/prohibitin/flotillin/HflK/C (SPFH) family that have been shown to influence membrane organization. Arabidopsis thaliana HIR2 interacts with multiple plasma membrane proteins, including receptor kinases such as BAK1-INTERACTING RECEPTORS 2 and 3 (BIR2 and 3), BRI1-ASSOCIATED KINASE 1 (BAK1), FLAGELLIN SENSING 2 (FLS2), and BRASSINOSTEROID INSENSITIVE 1 (BRI1). These interactions connect HIR2 to BAK1-mediated signaling pathways, as evidenced by impaired growth and immunity phenotypes in hir2 mutants. HIR2 is anchored to the inner leaflet of the plasma membrane through a hydrophobic interaction domain and S-acylation. Using single-particle tracking photoactivated localization microscopy (sptPALM), we showed that HIR2 affects receptor kinase dynamics and clustering, suggesting a role in spatially coordinating receptor complex activities. Structural modeling with AlphaFold 3 predicts a multimeric circular cup-like assembly for HIR2, consistent with high molecular weight complexes identified through blue native polyacrylamide gel electrophoresis. These findings indicate that HIR2 forms a discrete membrane compartment, providing a novel structural framework for spatial membrane organization and thereby modulating the function of membrane-resident receptors.
    Date: 2025-04-14
    Authors: Weber H, Ehinger A, Kolb D, Fallahzadeh-Mamaghani V, Halter T, Franz-Wachtel M, zur Oven-Krockhaus S, Gronnier J, Zipfel C, Harter K, Kemmerling B.
    Ref: bioRxiv
  12. Glioblastoma (GBM) is a common and highly lethal type of primary brain tumor in adults. Therapeutic failure is partly attributed to a fraction of Glioblastoma Stem Cells (GSCs) that show high levels of heterogeneity and plasticity. GSCs exist in a transcriptional gradient between two states: Developmental (Dev) and Injury Response (IR) in which IR-GSCs exhibit more invasive behaviors. While previous studies have identified fitness genes in GSCs, the genes required to establish and maintain the Dev and IR states remain poorly defined. To identify the regulators of the IR GSC state, we performed a phenotypic genome-wide CRISPR-Cas9 knockout (KO) screen in patient-derived GSCs based on cell surface expression of the IR marker CD44. Notably, we found that perturbations of the histone acetyltransferase EP300 in IR GSCs led to decreased CD44 cell surface expression, significant downregulation of gene expression signatures associated with the IR state, and to decreased self-renewal and invasion. Furthermore, genetic targeting of Ep300 in a mouse GBM model delayed tumor initiation and/or progression. Collectively, our results establish EP300 as a regulator of the IR state in GSCs and provide a mechanistic basis for its therapeutic targeting in GBM. Significance A genome-wide phenotypic CRISPR-Cas9 screen in a patient-derived Glioblastoma Stem Cell line identified the genes required to maintain the Injury-Response cellular state, with a focus on the histone acetyl transferase gene EP300 . This study suggests how therapeutic targeting of cellular state could reduce the aggressiveness of GBM tumors.
    Date: 2025-04-12
    Authors: Molaei F, MacLeod G, Haider S, Tiffany A, Oteng FM, Berman JM, Skirzynska A, Shoichet MS, Schramek D, Dirks PB, Angers S.
    Ref: bioRxiv
  13. Abstract Background The PELO–HBS1L complex is a critical mediator of ribosome-associated quality control, responsible for resolving stalled ribosomes and maintaining translational fidelity. Recent CRISPR-based synthetic lethality screens have identified PELO and HBS1L as selective vulnerabilities in cancers with 9p21.3 deletion or microsatellite instability-high (MSI-H) phenotypes. These cancers exhibit dependency on the PELO–HBS1L complex due to coexisting destabilization of the superkiller complex (SKIc), presenting a unique opportunity for targeted therapeutic intervention. However, no small-molecule inhibitors of this complex have been reported. Methods We employed AlphaFold2-multimer modeling to generate high-confidence structural models of the PELO–HBS1L heterodimer. Structural evaluation included predicted aligned error (PAE), per-residue confidence (pLDDT), and MSA coverage. Fragment-based pocket mapping identified a candidate binding pocket, selected for docking based on accessibility and proximity to inter-chain contacts. A fragment-guided design strategy was implemented using apogossypol scaffolds bearing hydroxyamide linkers and terminal polar groups. Ligands were prepared via Open Babel and docked into the interface pocket using AutoDock Vina. Binding energy, interaction geometry, and contact distances were used to rank binding poses. Results AlphaFold2 models revealed a rigid PELO core and a flexible HBS1L tail, with PAE and pLDDT scores indicating moderate interface confidence and ligand-accessible surface features. Indole-2-carboxylic acid with guanidine substitution formed key polar contacts in the candidate binding pocket. Among apogossypol analogs, methylamine and guanidine-tailed derivatives displayed strong polar interactions at the pocket interface, including engagement with GLU215, ASN, and THR. A methylated guanidine analog further enhanced interaction density, while piperidine substitutions were poorly tolerated. Conclusion A therapeutic small molecule docked in a binding pocket at the interface of two interacting proteins can disrupt a protein-protein complex. This approach, known as protein-protein interaction (PPI) inhibition, is a well-established strategy in drug discovery. The PELO–HBS1L interface contains a druggable surface pocket that can be targeted by small molecules. Apogossypol-derived ligands with flexible, cationic extensions demonstrate promising interaction profiles and provide a foundation for further hit-to-lead optimization. These findings support development of interface-directed inhibitors to exploit synthetic lethality in PELO-dependent cancers.
    Date: 2025-04-11
    Authors: Lehrer S, Rheinstein P.
    Ref: Research Square
  14. Abstract The oriental fruit fly Bactrocera dorsalis (Hendel) is considered as a quarantine pest in many countries and regions. Challenges remain in distinguishing this species with morphological similarities, especially in relevant development stages. In recent years, CRISPR-Cas12b genetic diagnostics has seen rapid advancements and offers an efficient tool for the identification of pathogens, viruses, and other genetic targets. Here we developed a new and rapid visual detection assay of B. dorsalis using recombinase polymerase amplification (RPA) and the CRISPR/Cas12b system. The system can detect different developmental stages of B. dorsalis within 30–35 minutes at 43 ℃ and the results are easily observed by the naked eye based on the color change in the tube during the reaction. The specificity and high sensitivity of this method was demonstrated, allowing for detection from 3.2 pg µL − 1 of DNA. With crude DNA, this diagnostic system successfully identified B. dorsalis by holding the reaction tubes in the hand. Our study demonstrates that RPA-CRISPR/Cas12b visualization system is effective to detect B. dorsalis rapidly and accurately. This approach can be applied for monitoring and identification of other pests in border and relevant locations, preventing biological invasions and ensuring pest control.
    Date: 2025-04-11
    Authors: Lv C, Zhang F, Ren L, Chen C, Zhu P, Cheng X, Yang X, Liu B.
    Ref: Research Square
  15. ABSTRACT Recently, de novo variants in an 18 nucleotide region in the centre of RNU4-2 were shown to cause ReNU syndrome, a syndromic neurodevelopmental disorder (NDD) that is predicted to affect tens of thousands of individuals worldwide 1,2 . RNU4-2 is a non-protein-coding gene that is transcribed into the U4 small nuclear RNA (snRNA) component of the major spliceosome 3 . ReNU syndrome variants disrupt spliceosome function and alter 5’ splice site selection 1,4 . Here, we performed saturation genome editing (SGE) of RNU4-2 to identify the functional and clinical impact of variants across the entire gene. The resulting SGE function scores, derived from variants’ effects on cell fitness, discriminate ReNU syndrome variants from those observed in the population and dramatically outperform in silico variant effect prediction. Using these data, we redefine the ReNU syndrome critical region at single nucleotide resolution, resolve variant pathogenicity for variants of uncertain significance, and show that SGE function scores delineate variants by phenotypic severity. Further, we identify variants impacting function in regions of RNU4-2 that are critical for interactions with other spliceosome components. We show that these variants cause a novel recessive NDD that is clinically distinct from ReNU syndrome. Together, this work defines the landscape of variant function across RNU4-2 , providing critical insights for both diagnosis and therapeutic development.
    Date: 2025-04-11
    Authors: De Jonghe J, Kim HC, Adedeji A, Leitão E, Dawes R, Chen Y, Blakes AJ, Simons C, Rius R, Alvi JR, Amblard F, Austin-Tse C, Baer S, Balton EV, Blanc P, Calame DG, Coutton C, Cunningham CA, Dargie N, Dipple KM, Du H, El Chehadeh S, Glass I, Gleeson JG, Grunewald O, Gueguen P, Harbuz R, Jacquemont M, Leventer RJ, Marijon P, Messaoud O, Sultan T, Thauvin C, Vincent-Delorme C, Yilmaz Gulec E, Thevenon J, Mendez R, MacArthur DG, Depienne C, Nava C, Whiffin N, Findlay GM.
    Ref: medRxiv
  16. The ribosome biases the conformations sampled by nascent polypeptide chains along folding pathways towards biologically active states. A hallmark of the co-translational folding (coTF) of many proteins are highly stable folding intermediates that are absent or only transiently populated off the ribosome, yet persist during translation well-beyond complete emergence of the domain from the ribosome exit tunnel. Intermediates are important for folding fidelity; however, their structures have remained elusive. Here, we have structurally characterised two coTF intermediates of an immunoglobulin-like domain by developing comprehensive 19 F NMR analyses using chemical shifts, paramagnetic relaxation enhancement (PRE), and protein engineering. We integrated these experimental data with extensive molecular dynamics (MD) simulations to obtain atomistic structures of the folding intermediates on the ribosome. The resulting intermediate structures are distinguished by native-like folds initiated from either their N-or C-termini, and reveal parallel folding pathways, which are structurally conserved within the protein domain family, in contrast to their in vitro refolding mechanisms. By redirecting proteins to fold along hierarchical, parallel routes, the ribosome may promote efficient folding by avoiding kinetic traps, and regulate nascent chain assembly and targeting by auxiliary factors to maintain cellular proteostasis.
    Date: 2025-04-11
    Authors: Chan SH, Streit JO, Włodarski T, Mitropoulou AN, Cassaignau AM, Bukvin IV, Cabrita LD, Christodoulou J.
    Ref: bioRxiv
  17. ABSTRACT Large scale loss-of-function screens utilising CRISPR or siRNA can provide profound insights into the importance of individual genes for the survival of a cancer cell and can drive the identification of therapeutic targets and biomarkers, and the development of targeted drugs. However, the analysis of these data and the substantial bodies of metadata that relate to them, is technically challenging and typically requires substantial expertise in data science and computer coding. To facilitate the analysis of cancer gene dependency data by cancer biologists and clinical scientists, we have developed DepMine – a computational toolkit providing a powerful system for framing complex queries relating cancer gene dependency to the underlying genetic changes that occur in cancer cells. DepMine identifies synthetic lethal relationships between putative target genes and complex ‘cancer profiles’ built from user-specified combinations of mutations, copy-number variation, and expression levels, and can refine these to optimal biomarker definitions for target dependency.
    Date: 2025-04-11
    Authors: Pearl LH, Pearl FM.
    Ref: bioRxiv
  18. SUMMARY While high and stable transgene expression can be achieved in undifferentiated pluripotent stem cells, conventional transgene expression systems are often silenced upon differentiation. Silencing occurs with both randomly integrated transgenes, introduced via transposase or lentiviral methods, and with transgenes targeted to specific genomic sites, including at commonly used safe harbor loci. The challenge to robustly express experimental transgenes in differentiated pluripotent stem cells is a major bottleneck in the field for applications such as CRISPR screening. Here, we conducted a comparative analysis to systematically evaluate the impact of various promoters, transcriptional regulatory elements, insulators, and genomic integration sites on transgene silencing during neuronal differentiation. Our findings reveal that specific combinations of promoters and transcriptional stability elements are able to prevent transgene silencing during differentiation, whereas chromatin insulators had less impact on silencing and three novel safe harbor integration sites performed similarly to the CLYBL locus. Guided by these insights we developed the PiggyBac vector TK4, which showed complete resistance to transgene silencing across various neuronal and microglial differentiation protocols from six different pluripotent stem cell lines, as independently confirmed by seven different laboratories. This construct will be highly useful for assays requiring stable transgene expression during differentiation, and holds the potential for broad applications in various research fields.
    Date: 2025-04-11
    Authors: Uenaka T, Napole AB, Saha AD, Sun D, Singavarapu A, Calzada L, Chen J, Erlebach L, McQuade A, Ramos D, Rigamonti A, Salazar L, Samelson AJ, Sedov K, Welsh NJ, Wild K, Wu Q, Arenas E, Bassett AR, Kampmann M, Kronenberg-Versteeg D, Merkle FT, Schüle B, Thompson LM, Skarnes WC, Ward ME, Wernig M.
    Ref: bioRxiv
  19. We present Alpha-BET, a structure-guided strategy leveraging AlphaFold to identify optimal ALFA-tag insertion sites for minimally disruptive labeling of viral glycoproteins with high-affinity nanobodies. Applied to HIV-1 Env, SARS-CoV-2 S, and NiV G, Alpha-BET preserves structural integrity and function. For HIV-1 Env, we demonstrate super-resolution DNA-PAINT MINFLUX 3D imaging enabled by tag insertion, showcasing its power for visualizing native trimers in single virions and potential for broader applications in virus research.
    Date: 2025-04-11
    Authors: Willliamson DJ, Zaza C, Carlon-Andres I, Gentili A, Daly JL, Holmes H, Thrush JW, Starling T, Neil S, Owens R, Malim MH, Tynan C, Simoncelli S, Padilla-Parra S.
    Ref: bioRxiv
  20. Abstract Genome editing by CRISPR-Cas9 is promising for genetic disease and cancer gene therapy. However, safety concerns are still present, particularly the ON-target genotoxicity for protocols using nucleases. Quality control of edited cells before and after graft is mandatory, especially to assay megabase-scale genomic rearrangements induced at the targeted locus. These unintended events are fortunately rare but potentially deleterious. Classical PCR-based bulk approaches do not detect them or underestimate their frequency. Single-cell approaches are promising but RNA sequencing only estimates copy number variation of the genome. Here, we propose single-cell DNA sequencing to accurately evaluate and monitor CRISPR-mediated genotoxicity in primary cells (human fibroblasts and hematopoietic stem/progenitor cells). We designed a homemade panel using single nucleotide polymorphisms to detect, map and characterize induced-losses of heterozygosity (terminal, interstitial, copy-loss and copy-neutral). This innovative approach revealed intense genotoxicity linked to the double strand break. The risk was associated with DNA repair modulators in particular DNA-PKcs inhibitor AZD7648. Importantly, the CDK4/6 inhibitor palbociclib prevented these rearrangements in hematopoietic stem/progenitor cells. This work strongly suggests that single-cell DNA sequencing should be routinely implemented in clinical applications before CRISPR-edited cell infusions.
    Date: 2025-04-11
    Authors: Moreau-Gaudry F, Boutin J, Fayet S, Marin V, BERGES C, Riandière M, Toutain J, Lamrissi-Garcia I, Thibault C, Cappellen D, Dabernat S, Poulet A, Francillette M, Droin N, Bedel A.
    Ref: Research Square
  21. Hemophilia B gene therapy treatments currently have not addressed the need for predictable, durable, active, and redosable factor IX (FIX). Unlike conventional gene therapy, engineered B Cell Medicines (BCMs) are durable, redosable, and titratable, and thus have the potential to address significant unmet needs in the Hemophilia B treatment paradigm. BE-101 is an autologous BCM comprised of expanded and differentiated B lymphocyte lineage cells genetically engineered ex vivo to secrete FIX-Padua. CRISPR/Cas9 mediated gene editing at the C-C chemokine receptor type 5 locus was used to facilitate transgene insertion of an AAV6-encoded DNA template via homology-directed repair. Transgene insertion did not alter B cell biology, viability, or differentiation into plasma cells. Appreciable levels of BE-101-derived FIX-Padua were detected within 1 day after IV administration in mouse and steady state was reached within 2 weeks and persisted for over 184 days. Redosing produced an increase in FIX-Padua production close to linear dose proportionality. Comprehensive genotoxicity analysis found no off-target issues of concern. No safety signals were observed in animal tolerability and GLP toxicology studies. In conclusion, BE-101 produces sustained levels of active FIX-Padua with the ability to engraft without host preconditioning and with the potential for redosing and titratability.
    Date: 2025-04-10
    Authors: Liu H, Singh S, Mullen TJ, Bullock C, Keegan S, Patterson T, Thakur S, Lundberg A, Shenker S, Couto R, Yadav C, Dastagir S, Li L, Bainter W, Liberzon E, Malloy CR, Lazzarotto CR, Ohsumi TK, Chilakala S, Chen H, Kshirsagar R, Hohmann AF, Arlauckas S, Lazorchak A, Scull C, Morgan RA.
    Ref: bioRxiv
  22. Simple Summary Diffuse midline gliomas (DMGs) are aggressive childhood brain tumours with no effective treatments. Over 80% of cases carry the histone H3K27M mutation, which alters chromatin structure and gene regulation and induce tumours growth. Immunotherapy, which uses the body’s immune system to fight cancer, relies on tumour- specific antigen molecules that immune cells can recognise. However, because DMGs have few mutations, finding suitable antigens for therapeutic purposes has been challenging. In this study, we investigated how the H3K27M mutation affects tumour antigen presentation in DMG. Using patient-derived DMG models, we found that H3K27M alters the landscape of antigens displayed on tumour cell surface, creating unique immune targets. We identified six immunogenic peptides, that triggered strong T cell responses. These antigens were absent when H3K27M was removed, confirming their link to the mutation. Our findings provide a blueprint for developing T cell-based immunotherapies for DMG, offering new hope for targeted treatments against this devastating disease. Background: Diffuse midline gliomas (DMGs) are among the most aggressive paediatric brain tumours, with the pathognomonic H3K27M mutation present in over 80% of cases. This mutation drives epigenetic dysregulation and transcriptional reprogramming, yet its impact on the tumour antigenic landscape remains poorly understood. Given the low mutational burden of DMG, an expanded search beyond neoantigens to include epigenetically dysregulated tumour-associated antigens (TAAs) is critical for advancing antigen-specific immunotherapies. Methods: To assess how H3K27M influences antigenic landscape of DMG, we performed a comprehensive immunopeptidomic analysis using patient-derived DMG cell line models (SU-DIPG13 and BT245) that harbour the H3K27M mutation and their CRISPR-edited H3K27M-knockout (KO) counterparts. High-resolution mass spectrometry and bioinformatics were employed to define H3K27M-driven changes in the immunopeptidome. Functional T cell assays using HLA-matched healthy donor PBMCs were conducted to evaluate the immunogenicity of H3K27M-associated peptides. Results: Our findings reveal that the H3K27M mutation reshapes the tumour antigenic landscape in a model-specific manner. While H3K27M knockout increased HLA-I expression in SU-DIPG13 but not BT245, immunopeptidomic profiling uncovered distinct shifts in the presentation of tumour-associated peptides, independent of direct effects on antigen processing machinery. Among these, we identified six immunogenic peptides, derived from SLITRK2, PRAME, XKR5, and CBX2, that elicited CD8⁺ T cell responses in in vitro functional assays. Notably, PRAME, a well-characterised cancer-testis antigen was confirmed as an H3K27M-associated immunogenic target, reinforcing its therapeutic relevance. Peptides identified exclusively in H3K27M+ cells were absent in KO models, demonstrating a direct link between H3K27M-driven transcriptional dysregulation and tumour antigenicity. Conclusions: This study provides the first systematic assessment of how H3K27M reshapes the antigenic landscape in DMG, uncovering novel, immunogenic tumour- associated peptides that could serve as targets for precision immunotherapy. By demonstrating that H3K27M mutation drives context-dependent antigen presentation, our findings establish a foundation for T cell-based therapies targeting H3K27M-associated antigens. These insights pave the way for next-generation personalised immunotherapies for this otherwise treatment-refractory disease. Key Points H3K27M mutation induces expression of tumour-associated antigens in DMG H3K27M alters the DMG immunopeptidome without uniformly changing HLA-I levels PRAME- and CBX2-derived peptides are immunogenic and targetable by CD8⁺ T cells Importance of Study Diffuse midline gliomas (DMGs) are universally fatal paediatric brain tumours with limited treatment options and poor immune visibility. While the H3K27M mutation is a defining hallmark, its impact on tumour immunogenicity remains unclear. This study presents the first comprehensive to explore the effect of H3K27M-mution on the DMG immunopeptidome, revealing six immunogenic peptides derived from epigenetically dysregulated tumour- associated antigens, including SLITRK2, PRAME, XKR5, and CBX2. These antigens elicited CD8⁺ T cell responses, establishing a direct link between H3K27M-driven transcriptional dysregulation and tumour antigenicity. By leveraging these altered antigens, we highlight actionable vulnerabilities for T cell-based immunotherapy.
    Date: 2025-04-10
    Authors: Shamekhi T, Zeng B, Sun CX, Daniel P, Kam Sian TCL, Goncalves G, Huang G, Fahimi F, Selvakumar N, Tanuwidjaya E, Woodhouse I, Kritzer B, Schittenhelm RB, Mazzieri R, Cain JE, Nazarin J, Hansford JR, Firestein R, Dolcetti R, Faridi P.
    Ref: bioRxiv
  23. Microgravity presents unique challenges to human physiology, particularly the immune system, during spaceflight. T lymphocytes, key components of adaptive immunity, play a vital role in immune regulation, yet the effects of microgravity on T-cell function and gene expression remain incompletely understood. This study, conducted as part of the MESSAGE (Microgravity Associated Genetics) Science Mission in the Axiom-3 mission ( https://www.nasa.gov/mission/station/research-explorer/investigation/?#id=9100 ). We aimed to investigate how microgravity influences immune cell responses by analysing blood samples from three astronauts between pre-flight and during their space mission on the ISS (post-launch Day 4, Day 7, and Day 10 in ISS). Hemogram analysis revealed no statistically significant differences in leukocyte, erythrocyte, haemoglobin, and haematocrit levels between pre-flight and in-flight samples, suggesting a stable haematological profile under microgravity conditions. T cell subpopulation analysis indicated fluctuations in effector memory T cells (TemEARLY and TemLATE), but these changes did not reach statistical significance. These findings suggest that, within the short duration of spaceflight, haematological and immune parameters remain largely stable, underscoring the need for further research into long-term immune adaptations in microgravity. As a next step, transcriptomic analysis will be performed to identify microgravity-associated gene candidates, and CRISPR-based knock-out of the genes in T cells will be generated to explore their functional roles in microgravity-induced immune modulation.
    Date: 2025-04-10
    Authors: Tastan C, Gulden G, Demir O, Cam E, Erkek F, Sert B, Gozler T, Yuksel I, Tekirdagli B.
    Ref: bioRxiv
  24. During their vectorial biosynthesis on the ribosome, elongating nascent polypeptide chains explore a range of conformational states towards their biologically functional structure. However, this high structural heterogeneity has limited their observation at high-resolution. Here, we have used an integrated structural biology approach to explore the structures of the multi-domain immunoglobulin-like FLN5-6 during its biosynthesis, capturing early folding through to native folding. We developed an in-silico purification approach for cryo-EM of ribosome-nascent chain complexes (RNCs), and integrated the resulting cryo-EM maps with NMR spectroscopy and atomistic molecular dynamics (MD) simulations to produce experimentally reweighted structural ensembles of RNC. The resulting atomistic structures reveal insights into the orientational heterogeneity of the nascent chain and its dynamic interactions with the ribosome. In particular, we find that two distinct pathways exist for nascent polypeptides in the exit tunnel vestibule, influenced by their stage of biosynthesis, folding conformational state and ribosomal RNA helices lining the tunnel. Our systematic analysis of the structures of nascent proteins translation-stalled at multiple time-points provides insights into how the ribosome dynamically modulates its pathway out of the exit tunnel to regulate its folding and accessibility for auxiliary factors of other co-translational events.
    Date: 2025-04-10
    Authors: Mitropoulou AN, Tomasz Włodarski T, Streit JO, Chan SH, Plessa E, Woodburn LF, Cabrita LD, Christodoulou J.
    Ref: bioRxiv
  25. ABSTRACT Human pluripotent stem cell (hPSC)-derived cardiac therapies hold great promise for heart regeneration but face major translational barriers due to allogeneic immune rejection. Here, we engineered hypoimmunogenic hPSCs using a two-step CRISPR-Cas9 strategy: (1) B2M knockout, eliminating HLA class I surface expression, and (2) knock-in of HLA-E or HLA-G trimer constructs in the AAVS1 safe harbor locus to confer robust immune evasion. Hypoimmunogenic hPSCs maintained pluripotency, efficiently differentiated into cardiac cell types that resisted both T and NK cell-mediated cytotoxicity in vitro , and self-assembled into engineered cardiac organoids. Comprehensive analyses of the hypoimmunogenic cells and organoids revealed preservation of transcriptomic, structural, and functional properties with minimal off-target effects from gene editing. In vivo , hypoimmunogenic cardiac organoids restored contractile function in infarcted rat hearts and demonstrated superior graft retention and immune evasion in humanized mice compared to wild-type counterparts. These findings establish the therapeutic potential of hypoimmunogenic hPSC-CMs in the cardiac organoid platform, laying the foundation for off-the-shelf cardiac cell therapies to treat cardiovascular disease, the leading cause of death worldwide.
    Date: 2025-04-09
    Authors: Silver SE, Howells AR, Arhontoulis DC, Randolph LN, Hyams NA, Barrs RW, Li M, Kerr CM, Robino RA, Morningstar JE, Bain JD, Floy ME, Norris RA, Bao X, Ruddy JM, Palecek SP, Ferreira LMR, Lian XL, Mei Y.
    Ref: bioRxiv
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