Results 851-875 of about 1,000
  1. Mitochondria control cellular metabolism, serve as hubs for signaling and organelle communication, and are important for the health and survival of cells. _VPS13D_ encodes a cytoplasmic lipid transfer protein that regulates mitochondrial morphology, mitochondria and endoplasmic reticulum (ER) contact, quality control of mitochondria. _VPS13D_ mutations have been reported in patients displaying ataxic and spastic gait disorders with variable age of onset. Here we used CRISPR/Cas9 gene editing to create _VPS13D_ related-spinocerebellar ataxia-4 (SCAR4) missense mutations and C-terminal deletion in _VPS13D_’s orthologue _vps-13D_ in _C. elegans_. Consistent with SCAR4 patient movement disorders and mitochondrial dysfunction, _vps-13D_ mutant worms exhibit locomotion defects and abnormal mitochondrial morphology. Importantly, animals with a _vps-13D_ deletion or a N3017I missense mutation exhibited an increase in mitochondrial unfolded protein response (UPRmt). The cellular and behavioral changes caused by _VPS13D_ mutations in _C. elegans_ advance the development of animal models that are needed to study SCAR4 pathogenesis.
    Date: 2025-02-14
    Authors: Wang R, Thackeray A, Baehrecke EH, Alkema MJ.
    Ref: Qeios
  2. ABSTRACT LMNA -associated congenital muscular dystrophy is a currently incurable rare genetic disorder characterized by early-onset muscle weakness, dilated cardiomyopathy and respiratory failure, resulting from mutations in the LMNA gene. In this study, we assessed the potential of a CRISPR-mediated strategy to eliminate the mutant allele Lmna c.745C>T, p.R249W using a mutation specific guide (sg745T). Results from R249W-mutation-carrying cellular models showed specific activity of the Cas9/sg745T complex towards the mutant allele. This property varied depending on the concentration of CRISPR components, with a loss of specificity observed with increased dosage. We tested this strategy in vivo using adeno-associated virus delivery in Lmna R249W mice. Despite being associated with a modest CRISPR activity, this therapeutic approach resulted in a 10% increase in the survival of R249W homozygous mice. Interestingly, a similar CRISPR activity improved the cardiac pathology developed by Lmna +/R249W animals, significantly extending their median survival. These results represent the first therapeutic validation of a CRISPR/Cas9-mediated gene editing strategy for the treatment of LMNA -associated congenital muscular dystrophy.
    Date: 2025-02-14
    Authors: Gómez-Domínguez D, Epifano C, Hernández I, Vilaplana-Martí B, Cesar S, de Molina-Iracheta A, Sena-Esteves M, Sarquella-Brugada G, Pérez de Castro I.
    Ref: bioRxiv
  3. T cells play a crucial role in the adaptive immune system and depend on tightly regulated intracellular signalling pathways to respond in an appropriate manner. Adapter proteins have flexible and dynamic features, which allow them to regulate T cell signal transduction pathways. As adapter proteins are enzymatically inert and may play multiple roles in parallel, it has been a challenge to fully characterise their functions individually. One such protein in T cells, is T cell specific adapter protein (TSAd), which is upregulated following T cell receptor (TCR) stimulation and is believed to mediate Src family tyrosine kinase signalling. However, the functional role remains elusive, possibly due to limited insight into interactors that potentially bind TSAd. The only structurally well-defined feature within TSAd, is the Src homology 2 (SH2) domain. This conserved domain displays prototypic binding of phosphorylated tyrosines, which suggests that the adapter molecule is implicated in phosphotyrosine signalling pathways. Here, we used an unbiased approach to identify ligands of the TSAd SH2 domain, by using affinity-purification mass spectrometry (AP-MS). Several novel ligands, many of which are known to be implicated in negative regulation of T cell intracellular signalling, were identified. More specifically, we showed that TSAd binds DOK2 and PTPN11 and determined the tyrosines responsible for the TSAd SH2 domain-dependent interaction. Ablation of TSAd and DOK2 by CRISPR/Cas9 in Jurkat T cells resulted in altered tyrosine phosphorylation. Taken together, these findings provide new insight into the possible function of TSAd as a negative signalling node in T cells.
    Date: 2025-02-14
    Authors: Chan H, Borowicz P, Gilmour BC, Stensland M, Garcia-Loza I, Phuyal S, Marone R, Jeker LT, Nyman TA, Spurkland A.
    Ref: bioRxiv
  4. ABSTRACT Existing CRISPR-based genome editing tools are limited in Bacillus subtilis due to the large cas gene. The recently reported DNA nuclease IscB has the potential to be developed into a novel genome editing tool due to its size being one-third of Cas9, while its application in B. subtilis remains unexplored. In this study, genome editing tools pBsuIscB/pBsuenIscB based on IscB and enIscB (enhanced IscB) were established in B. subtilis SCK6, and successfully deleted 0.6 kb to 4.3 kb genes with efficiencies up to 100%. Subsequently, the pBsuenIscB with higher deletion efficiency was used, whereby the large genomic fragment of 37.7 kb or 169.9 kb was deleted with only one ωRNA. Additionally, single-copy or multi-copy mCherry genes was integrated by using pBsuenIscB. Finally, the editing plasmid was eliminated and the second round of genome editing was completed. Overall, this study has successfully applied IscB to B.subtilis , expanded the genome editing toolbox of B. subtilis , and will help to construct B. subtilis chassis for production of a variety of biomolecules.
    Date: 2025-02-14
    Authors: Gao J, Tang H, Yang Y, Wang H, Li Q.
    Ref: bioRxiv
  5. Abstract OsbZIP35 is a member of the B subfamily of bZIP transcription factors in rice ( Oryza sativa L.). The function of OsbZIP35 has not been reported previously. In this study, drought, H 2 O 2 , abscisic acid, and NaCl treatments strongly induced the expression of OsbZIP35 , whereas treatment with gibberellin, indoleacetic acid, and jasmonic acid did not affect OsbZIP35 expression. We used the CRISPR/Cas9 gene-editing technology to construct OsbZIP35 knockout mutants ( bzip35-1 and bzip35-2 ) and evaluated the function of OsbZIP35 . The results showed that, compared with the wild type, the bzip35 mutants were more sensitive to drought stress during the germination, post-germination growth, and seedling stages. Specifically, the bzip35 mutants exhibited a lower germination percentage, a weaker growth phenotype, and an increase in reactive oxygen species accumulation. Further analysis indicated that, under drought stress, OsbZIP35 regulated reactive oxygen species accumulation by modulating the contents of antioxidants, thereby positively regulating the response of rice seedlings to drought stress. In addition, we observed that OsbZIP35 could be phosphorylated by OsSAPK3 and participated in the abscisic acid signaling pathway in response to drought stress. Agronomic trait analysis revealed that, under drought stress at the heading stage, the panicle length and seed-set rate of bzip35 mutants were significantly lower than those of the wild type. This study examined the role of OsbZIP35 in rice stress tolerance and yield regulation, and identified an upstream regulatory gene, OsSAPK3 . The results provide novel information on the mechanisms of stress tolerance and yield regulation in rice.
    Date: 2025-02-13
    Authors: Lou D, Zhang X, Chen Z, Wang Y, Yu D, Yang X.
    Ref: Research Square
  6. Our perception of the world depends on the brain’s ability to integrate information from multiple senses, with temporal disparities providing a critical cue for binding or segregating cross-modal signals 1,2 . The superior colliculus (SC) is a key site for integrating sensory modalities, but how cellular and network mechanisms in distinct anatomical regions within the SC contribute to multisensory integration remains poorly understood. Here, we recorded responses from over 5,000 neurons across the SC’s anatomical axes of awake mice during presentations of spatially coincident audiovisual stimuli with varying temporal asynchronies. Our findings revealed that multisensory neurons reliably encoded audiovisual delays and exhibited nonlinear summation of auditory and visual inputs, with nonlinearities being more pronounced when visual stimuli preceded auditory stimuli, consistent with the natural statistics of light and sound propagation. Nonlinear summation was crucial for population-level decoding accuracy and precision of AV delay representation. Moreover, enhanced population decoding of audiovisual delays in the posterior-medial SC, facilitated temporal discriminability in the peripheral visual field. Cross-correlation analysis indicated higher connectivity in the medial SC and functional specific recurrent connectivity, with visual, auditory, and multisensory neurons preferentially connecting to other neurons of the same functional subclass, and multisensory neurons receiving approximately 50 percent of the total local input from other multisensory neurons. Our results highlight the interplay between single-neuron computations, network connectivity, and population coding in the SC, where nonlinear integration, distributed representations and regional functional specialisations enables robust sensory binding and supports the accurate encoding of temporal multisensory information. Our study provides new insights into how the brain leverages both single-neuron and network-level mechanisms to represent sensory features by adapting to the statistics of the natural world.
    Date: 2025-02-12
    Authors: Bianchini G, Razafindrahaba I, Moglie MJ, Konstantinou G, Cano-Ferrer X, Imbert A, Iacaruso MF.
    Ref: bioRxiv
  7. The ability to identify gene functions and interactions in specific cellular contexts has been greatly enabled by functional genomics technologies. CRISPR-based genetic screens have proven invaluable in elucidating gene function in mammalian cells. Single-cell functional genomics methods, such as Perturb-seq and Spear-ATAC, have made it possible to achieve high-throughput mapping of the functional effects of gene perturbations by profiling transcriptomes and DNA accessibility, respectively. Combining single-cell chromatin accessibility and transcriptomic data via multiomic approaches has facilitated the discovery of novel cis and gene regulatory interactions. However, pseudobulk readouts from cell populations can often cloud the interpretation of results due to a heterogeneous response from cells receiving the same genetic perturbation, which could be mitigated by using transcriptional profiles of single cells to subset the ATAC-seq data. Existing methods to capture CRISPR guide RNAs to simultaneously assess the impact of genetic perturbations on RNA and ATAC profiles require either cloning of gRNA libraries in specialized vectors or implementing complex protocols with multiple rounds of barcoding. Here, we introduce CAT-ATAC, a technique that adds CRISPR gRNA capture to the existing 10X Genomics Multiome assay, generating paired transcriptome, chromatin accessibility and perturbation identity data from the same individual cells. We demonstrate up to 77% guide capture efficiency for both arrayed and pooled delivery of lentiviral gRNAs in induced pluripotent stem cells (iPSCs) and cancer cell lines. This capability allows us to construct gene regulatory networks (GRNs) in cells under drug and genetic perturbations. By applying CAT-ATAC, we were able to identify a GRN associated with dasatinib resistance, indirectly activated by the HIC2 gene. Using loss of function experiments, we further validated that the gene, ZFPM2, a component of the predicted GRN, also contributes to dasatinib resistance. CAT-ATAC can thus be used to generate high-content multidimensional genotype-phenotype maps to reveal novel gene and cellular interactions and functions.
    Date: 2025-02-12
    Authors: Shevade K, Yang YA, Feng K, Mader K, Sevim V, Parsons J, Arora G, Elfawy H, Mace R, Federman S, Esanov R, Shafer S, Chow ED, Przybyla L.
    Ref: bioRxiv
  8. Genome editing enzymes can introduce targeted changes to the DNA in living cells 1–4 , transforming biological research and enabling the first approved gene editing therapy for sickle cell disease 5 . However, their genome-wide activity can be altered by genetic variation at on- or off-target sites 6–8 , potentially impacting both their precision and therapeutic safety. Due to a lack of scalable methods to measure genome-wide editing activity in cells from large populations and diverse target libraries, the frequency and extent of these variant effects on editing remains unknown. Here, we present the first population-scale study of how genetic variation affects the cellular genome-wide activity of CRISPR-Cas9, enabled by a novel, sensitive, and unbiased cellular assay, GUIDE-seq-2 with improved scalability and accuracy compared to the original broadly adopted method 9 . Analyzing Cas9 genome-wide activity at 1,115 on- and off-target sites across six guide RNAs in cells from 95 individuals spanning four genetically diverse populations, we found that variants frequently overlap off-target sites, with 13% significantly altering Cas9 editing activity by up to 33% indels. To understand common features of high-impact variants, we developed a new massively parallel biochemical assay, CHANGE-seq-R, to measure Cas9 activity across millions of mismatched target sites, and trained a deep neural network model, CHANGE-net, to accurately predict and interpret the effects of single-nucleotide variants on off-targets with up to six mismatches. Taken together, our findings illuminate a path to account for genetic variation when designing genome editing strategies for research and therapeutics.
    Date: 2025-02-12
    Authors: Lazzarotto CR, Li Y, Flory AR, Chyr J, Yang M, Katta V, Urbina E, Lee G, Wood R, Matsubara A, Rashkin SR, Ma J, Cheng Y, Tsai SQ.
    Ref: bioRxiv
  9. Mismatch repair (MMR) is a crucial DNA repair pathway that maintains genomic integrity by correcting replication errors and various forms of DNA damage. MMR deficiency (MMRd) leads to increased mutation rates, microsatellite instability, and contributes to tumorigenesis in multiple cancer types. Using a CRISPR-Cas9-mediated knockout assay in human isogenic cell lines, we characterised mutational profiles in MMR-deficient cells. Our findings revealed expected increases in mutation burden and the emergence of known MMR-associated mutational signatures. Notably, we identified a previously unconnected process, SBS57, and linked it to germline single-nucleotide polymorphisms and MMR-driven indels in MMRd cells, establishing its association with tensor signature TS27. Comparative analyses of in vitro MMRd profiles and in vivo tumour data uncovered key differences in mutational signatures, highlighting the biological context dependence of MMR-associated mutations. Furthermore, we provide direct experimental evidence that MMR plays a role in repairing 5-methylcytosine deamination, a repair process previously inferred from tumour sequencing data. These findings offer novel insights into MMR deficiency, shedding light on previously uncharacterised mutational mechanisms and their implications in cancer.
    Date: 2025-02-12
    Authors: Owusu M, Menche J, Loizou J, Weghorn D.
    Ref: bioRxiv
  10. Photosynthesis is a biological process that converts light energy into chemical energy. Excessive light can damage the photosynthetic machinery, so plants have evolved photoprotective mechanisms such as non-photochemical quenching (NPQ). Among the NPQ mechanisms, qH is a form of sustained quenching, dependent on LIPOCALIN IN THE PLASTID (LCNP) and repressed by SUPPRESSOR OF QUENCHING 1 (SOQ1), protecting against abiotic stress. Recently, we showed in Arabidopsis thaliana that qH can occur in the major light-harvesting complexes (Lhcb1, Lhcb2, Lhcb3) but independently of any specific major antenna. Interestingly, in mutants with little or no accumulation of major antennae ( koLHCII, lhcb1, cpsrp43 ), qH can still be induced. Here, we show that the minor antennae can be quenched by qH and remain quenched once isolated. To investigate the role of minor antennae in qH, we combined the soq1 mutant, which displays high qH, with mutations in each minor antenna type (Lhcb4, Lhcb5, or Lhcb6), or with a mutant lacking all minor antennae. None are strictly required for qH to occur. Still, the absence of Lhcb6 decreases qH induction likely due to an indirect effect from the slower electron transport rate and/or a different macro-organization of photosynthetic complexes in the thylakoids. Overall, this work demonstrates that the minor antennae are a secondary target for qH and could serve as an additional safety valve for photoprotective energy dissipation during prolonged stress.
    Date: 2025-02-12
    Authors: Bru P, Crepin A, Provot Y, Guardini Z, Bassi R, Dall’Osto L, Malnoë A.
    Ref: bioRxiv
  11. SUMMARY Compelling evidence demonstrates a functional link between neuronal activity and myelination, highlighting the vital importance of axon-oligodendrocyte crosstalk in myelin physiology and function. However, how neuronal activity is relayed to oligodendroglia to regulate myelin formation remains not fully understood. Here, we aimed to characterize how that myelination is regulated by glutamate vesicular release in zebrafish spinal cord. We compared oligodendrocyte precursor cells (OPCs) and myelinating oligodendrocytes (mOLs) for their close apposition with pre-synaptic boutons and found that these are increased in number on mOLs during myelin internode elongation. Consistently, mOLs show more pre-synaptic boutons during myelin internode elongation compared to OPCs. In addition, we also found that oligodendroglial cells express the post-synaptic density protein 95 (PSD-95) along punctated domains, regardless of their differentiation stage. Genetically targeted PSD-95-GFP expression in oligodendroglia revealed post-synaptic-like domains along their processes and sheaths, which are contacted by axonal pre-synaptic varicosities. These contacts are increased in mOLs. Importantly, CRISPR-Cas9 mediated deletion of dlg4 in oligodendroglia impairs myelin sheath growth , in vivo . Overall, our data indicate that PSD-95 is a key component of axons to oligodendrocytes neurotransmission that regulates myelin sheath growth. HIGHLIGHTS Glutamate vesicular release is required for myelination Axon-oligodendroglia connectivity increases with oligodendrocyte maturation Oligodendrocytes express the post-synaptic density protein 95 Dlg4 loss-of-function in oligodendroglia impedes myelin sheath growth GRAPHICAL ABSTRACT
    Date: 2025-02-11
    Authors: Masson M, Graciarena M, Porte M, Nait Oumesmar B.
    Ref: bioRxiv
  12. Induced pluripotent stem cell (iPSC) models are powerful tools for neurodegenerative disease modelling, as they allow mechanistic studies in a human genetic environment and they can be differentiated into a range of neuronal and non-neuronal cells. However, these models come with inherent challenges due to line-to-line and clonal variability. To combat this issue, the iPSC Neurodegenerative Disease Initiative (iNDI) has generated an iPSC repository using a single clonal reference line, KOLF2.1J, into which disease-causing mutations and revertants are introduced via gene editing. Here we describe the generation and validation of lines carrying the most common causative mutation for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), a repeat expansion in the C9orf72 gene, for the iNDI collection of neurodegenerative iPSC models. We demonstrate that these C9orf72 knock-in lines differentiate efficiently into neurons and display characteristic C9orf72 -associated pathologies, including reduced C9orf72 levels and the presence of dipeptide repeat proteins (DPRs) and RNA foci, which increase in abundance over time in culture. These pathologies are not present in revertant cells lacking the repeat expansion. These repeat expansion and revertant cell lines are now available to academic and for-profit institutions through the JAX iPS cell repository and will help to facilitate and standardise iPSC-based ALS/FTD research.
    Date: 2025-02-11
    Authors: Coneys R, Cammack AJ, Nair RR, Thompson D, Mechtersheimer J, Carcolé M, Gupta Y, Rech GE, Flower M, O’Brien N, Ruepp M, Mizielinska S, Ducotterd F, Tabrizi SJ, Fisher EM, Cunningham TJ, Ward M, Skarnes WC, Isaacs AM.
    Ref: bioRxiv
  13. Objective Candida auris has emerged as a fungal pathogen of particular concern owing in part to its propensity to exhibit antifungal resistance, especially to the commonly prescribed antifungal fluconazole. In this work we aimed to determine how mutations in the transcription factor gene TAC1B , which are common among resistant isolates and confer fluconazole resistance, exert this effect. Methods Selected TAC1B mutations from clinical isolates were introduced into a susceptible isolate and reverted to the wild-type sequence in select clinical isolates using CRISPR Cas9 gene editing. Disruption mutants were likewise generated for select genes of interest. TAC1B mutants were subjected to transcriptional profiling by RNA-seq, and relative expression of specific genes of interest was determined by qRT-PCR. Antifungal susceptibilities were determined by modified CLSI broth microdilution. Results TAC1B mutations leading to A640V, A657V, and F862_N866del conferred fluconazole resistance, as well as increased resistance to other triazoles, when introduced into a susceptible isolate. RNA-seq revealed that the ATP-Binding Cassette (ABC) transporter gene CDR1 as well as the Major Facilitator Superfamily (MFS) transporter gene MDR1 were both upregulated by these TAC1B mutations. Disruption of CDR1 greatly abrogated resistance in strains with TAC1B mutations whereas disruption of MDR1 had little to no effect. However, disruption of both CDR1 and MDR1 resulted in an additional reduction in resistance as compared to disruption of either gene alone. Conclusion TAC1B mutations leading to A640V, A657V, and F862_N866del all result in increased resistance to fluconazole and other triazole antifungals, and increased expression of both CDR1 and MDR1 in C. auris . CDR1 is the primary driver of resistance conferred by these TAC1B mutations.
    Date: 2025-02-11
    Authors: Barker KS, Santana DJ, Zhang Q, Peters TL, Rybak JM, Morschhäuser J, Cuomo CA, Rogers PD.
    Ref: bioRxiv
  14. Kaposi Sarcoma-associated herpesvirus (KSHV) persists as a latent episome in infected cells. While the virus efficiently infects established cell lines and primary cells in vitro , the early events guiding establishment of latent infection and the dynamic interplay between viral episomes and host factors remain incompletely understood. Here, we describe the development and application of a CRISPR/Cas9-based 3D live cell imaging system capable of tracking single KSHV episomes in real-time. Our approach exploits the SunTag technology, wherein deactivated Cas9 (dCas9) molecules are fused to repetitive epitope arrays recognized by superfolder GFP-fused single-chain antibodies. By targeting these complexes to terminal repeat units of KSHV, we achieve high level signal amplification, allowing us not only to detect newly incoming viral genomes within the first hours of de novo infection, but also to follow their spatiotemporal trajectories through different stages of the viral lifecycle. Furthermore, to facilitate efficient generation of stable reporter cell lines, we adapted the transposon-based piggyBac system to combine all SunTag components into a single-vector targeting system (SunSeT). Using these systems, we demonstrate the ability to observe both transient and stable interactions between KSHV episomes and key cellular regulators, including the variant polycomb-repressive complex 1 (vPRC) component KDM2B and the innate immune sensor IFI16. Furthermore, our platform allows detailed visualization of episodic changes in episome localization, abundance and distribution during de novo and long-term infection, providing critical insights into how viral genome positioning and dynamics correlate with host subnuclear environments. Overall, our study introduces a robust and adaptable imaging platform to dissect the earliest events of KSHV infection. The ability to track viral episomes in living cells offers a powerful tool to advance our understanding of the spatial and temporal regulation of individual KSHV genomes, shedding light on fundamental mechanisms of herpesvirus latency and persistence.
    Date: 2025-02-11
    Authors: Günther T, Weissmann S, Hamann MV, Scheibel H, Bosse JB, Ziegler M, Grundhoff A.
    Ref: bioRxiv
  15. Phenotypic outcomes can be heavily affected by environmental factors. In this study, we exploited the previously observed nutrient-dependency of cell biological phenotypic features, captured by a cross-condition image-based profiling assay of Escherichia coli deletion strains, to examine this in more detail. We identified several general principles, including the existence of a spectrum of deviating phenotypes across nutrient conditions (i.e., from nutrient- or feature-specific to pleiotropic phenotypic deviations), limited conservation of phenotypic deviations across nutrient conditions (i.e., limited phenotypic robustness), and a subset of nutrient-independent phenotypic deviations (indicative of consistent genetic determinants of specific phenotypic features). In a subsequent step, we used this cross-condition dataset to identify five genes of unknown function of which the deletion displayed either nutrient-independent phenotypic deviations or phenotypic similarities to genes of known function: yibN , yaaY , yfaQ , ybiJ , and yijD . These genes showed different levels of phylogenetic conservation, ranging from conserved across the tree of life ( yibN ) to only present in some genera of the Enterobacterales ( yaaY ). Analysis of the structural properties of the proteins encoded by these y-genes, identification of structural similarities to other proteins, and the examination of their subcellular localization yielded new insights into their contribution to E. coli cell morphogenesis, cell cycle progression and cell growth. Together, our approach showcases how bacterial image-based profiling assays and datasets can serve as a gateway to reveal the function of uncharacterized proteins. Importance Despite unprecedented access to genomic information, predicting phenotypes based on genotypes remains notoriously difficult. One major confounding factor is the environment and its ability to modulate phenotypic outcomes. Another is the fact that a large fraction of protein-coding genes in bacterial genomes remains uncharacterized and have no known function. In this work, we use a large-scale cross-condition image-based profiling dataset to characterize nutrient-dependent phenotypic variability of E. coli deletion strains and exploit it to provide insight into the cellular role of genes of unknown function. Through our analysis, we identified five genes of unknown function that we subsequently further characterized by examining their phylogenetic conservation, predicted structural properties and similarities, and their intracellular localization. Combined, this approach highlights the potential of cross-condition image-based profiling, which extracts many cell biological phenotypic readouts across multiple conditions, to better understand nutrient-dependent phenotypic variability and uncover protein function.
    Date: 2025-02-11
    Authors: Sondervorst K, Nesporova K, Herdman M, Steemans B, Rosseels J, Govers SK.
    Ref: bioRxiv
  16. ABSTRACT Oncogenic gene fusions are key drivers of cancer, yet most remain untargetable by current therapies. Here, we establish CRISPR- Psp Cas13b as a personalizable platform for systematic silencing of various fusion transcripts. We reveal that recognition and cleavage of the breakpoint sequence by PspCas13b disrupts the fusion transcript, resulting in unexpected RNA nicking and ligation near the cleavage site, which generates out-of-frame, translation-incompetent transcripts. This approach efficiently degrades canonical and drug-resistant BCR::ABL1 mutants (e.g., T315I), a primary cause of resistance to tyrosine kinase inhibitors (TKIs) and relapse in chronic myeloid leukemia (CML). Silencing T315I BCR::ABL1 mRNA in drug-resistant CML cells triggers extensive transcriptomic, proteomic, and phosphoproteomic remodelling, causing erythroid differentiation and apoptosis. Beyond BCR-ABL1 mutants, personalized design of Psp Cas13b effectively silences other undruggable fusions, including RUNX1::RUNX1T1 and EWSR1::FLI1, key drivers in acute myeloid leukemia and in Ewing sarcoma, respectively. Collectively, this study establishes a framework for systematic, precise, and personalizable targeting of otherwise undruggable or drug-resistant oncogenic transcripts.
    Date: 2025-02-11
    Authors: Hu W, Chen H, Casan JM, Shembrey C, He S, Brown LM, Hughes TP, White DL, De Weck A, Voskoboinik I, Trapani JA, Ekert PG, Sadras T, Fareh M.
    Ref: bioRxiv
  17. ABSTRACT Regulation of protein synthesis is central to maintaining skeletal muscle integrity and its understanding is important for the treatment of muscular and neuromuscular pathologies. The eIF3f subunit of the translation initiation factor eIF3 has a key role, as it stands at the crossroad between protein-synthesis-associated hypertrophy and MAFbx/atrogin-1-dependent. To decipher the molecular mechanisms underpinning the role of eIF3f in regulating muscle mass, we established a cellular model that enables interrogation of eIF3f functionality via identification of proximal interactors. Using CRISPR-Cas9 molecular scissors, we generated single cell clones of immortalised human muscle cells expressing eIF3f fused to the BirA biotin ligase (eIF3f-BioID1 chimera) from the endogenous EIF3F locus. Biotinylated proteins, representing interactors of eIF3f in nanometer range distance, were identified by streptavidin pull-downs and mass spectrometry. In both proliferating and differentiated muscle cells, the eIF3f-BioID1 chimera co-sedimented with ribosomal complexes in polysome profiles and interacted mainly with components of the eIF3 complex, and with the eIF4E, eIF4G, and eIF5 initiation factors. Surprisingly, we identified several nucleus-localised interactors of eIF3f, and the immunofluorescence analyses revealed a previously unknown nuclear localization of eIF3f in both myoblasts and myotubes. We also identified novel cytoplasmic partners of eIF3f, responsible for the maintenance of skeletal muscle ultrastructure (sarcomeric/Z-disc (SYNPO2) bound proteins) and proteins of the lysosomal compartment (LAMP1). The established tagging system should be useful to further advance studies of eIF3f function in hypertrophic and atrophic conditions in skeletal muscle.
    Date: 2025-02-10
    Authors: Tintignac L, Mittal N, Alam S, Ataman M, Ertuna YI, Bock T, Erne B, Zavolan M, Sinnreich M.
    Ref: bioRxiv
  18. Abstract Endocrine therapy in combination with CDK4/6 inhibition doubles the progression-free survival of patients with advanced ER + breast cancer, but resistance is inevitable, leaving patients with limited treatment options. Here, we performed unbiased genome-wide CRISPR/Cas9 knockout screens using ER + breast cancer cells to identify novel drivers of resistance to combination endocrine therapy (tamoxifen) and CDK4/6 inhibitor (palbociclib) treatment. Our screens identified the inactivation of JNK signalling, including loss of the kinase MAP2K7 , as a key driver of combination resistance. We developed multiple CRISPR/Cas9 knockout ER + breast cancer cell lines (MCF-7 and T-47D) to investigate the effects of MAP2K7 and downstream MAPK8 and MAPK9 loss. MAP2K7 knockout increased metastatic burden in vivo and led to impaired JNK-mediated stress responses, as well as promoting cell survival and reducing senescence entry following endocrine therapy and CDK4/6 inhibitor treatment. Mechanistically, this occurred via loss of the AP-1 transcription factor c-JUN, leading to an attenuated response to combination endocrine therapy plus CDK4/6 inhibition. Furthermore, we analysed ER + advanced breast cancer patient cohorts and found that inactivation of the JNK pathway was associated with increased metastatic burden, and low pJNK T183/Y185 activity correlated with a poorer response to systemic endocrine and CDK4/6 inhibitor therapies. Overall, we demonstrate that suppression of JNK signalling enables persistent growth during combined endocrine therapy and CDK4/6 inhibition. Our data provide a pre-clinical rationale to screen patients’ tumours for JNK signalling deficiency prior to receiving combined endocrine therapy and CDK4/6 inhibition.
    Date: 2025-02-10
    Authors: Caldon C, Alexandrou S, Lee C, Fernandez K, Wiharja C, Eshraghi L, Reeves J, Reed D, Portman N, Phan Z, Milioli H, Nikolic I, Cadell A, Croucher D, Simpson K, Lim E, Hickey T, Millar E, Alves C, Ditzel H.
    Ref: Research Square
  19. Parasitic plants initiate rapid de novo organogenesis of a specialized feeding structure called a haustorium upon contact with their hosts. Currently, little is known about the internal signals regulating haustorium development. Here, we identify root meristem growth factor (RGF) peptides in Phtheirospermum japonicum as endogenous inducers of prehaustorium formation. Treatment with specific RGF peptides in the absence of hosts triggered prehaustoria and induced expression of PjYUC3 , a gene required for auxin biosynthesis and prehaustorium formation. CRISPR-mediated knockouts showed that PjRGFR1 and PjRGFR3, receptors activated by the haustorium specific RGF peptides PjRGF2 and PjRGF5, are essential for prehaustorium formation, revealing functional redundancy. Phylogenic analyses indicate that PjRGF2 is broadly conserved among Orobanchaceae, whereas PjRGF5 appears to have recently evolved through tandem multiplication and neofunctionalization. Our findings establish RGF peptides and their corresponding receptors as critical components of haustorium developmental signaling and provide insights into the evolutionary trajectories that shape plant parasitism. Teaser Plant peptide hormones regulate and induce the parasitic plant specialized organ for connecting to and feeding from the host.
    Date: 2025-02-10
    Authors: Fishman MR, Greifenhagen A, Wakatake T, Laohavisit A, Hiroyama R, Masuda S, Shibata A, Yoshida S, Shirasu K.
    Ref: bioRxiv
  20. ABSTRACT Coronaviruses, including SARS-CoV-2, rely on host factors for their replication and pathogenesis, while hosts deploy defense mechanisms to counteract viral infections. Although numerous host proviral factors have been identified, the landscape of host restriction factors and their underlying mechanisms remain less explored. Here, we conducted genome-wide CRISPR knockout screens using three distinct coronaviruses—SARS-CoV-2, HCoV-OC43 (a common cold human virus from the genus Betacoronavirus ) and porcine epidemic diarrhea virus ( Alphacoronavirus ) to identify conserved host restriction factors. We identified glycosylphosphatidylinositol (GPI) biosynthesis as the pan-coronavirus host factor that restrict viral entry by disrupting spike protein-mediated membrane fusion at both endosomal and plasma membranes. GPI biosynthesis generates GPI moieties that covalently anchor proteins (GPI-anchored proteins [GPI-APs]) to the cell membrane, playing essential roles in various cellular processes. Through focused CRISPR knockout screens targeting 193 GPI-APs, we identified LY6E as the key downstream effector mediating the antiviral activity of the GPI biosynthesis pathway. These findings reveal a novel role for GPI biosynthesis as a conserved host defense mechanism against coronaviruses and highlight LY6E as a critical antiviral effector. This study provides new insights into virus-host interactions and the development of host-directed antiviral therapies.
    Date: 2025-02-10
    Authors: Ma Y, Feng F, Feng H, Ma X, Wang Z, Han Y, Zhu Y, Wang Y, Gao Z, Zhang Y, Zhao J, Zhang R.
    Ref: bioRxiv
  21. The regulation of mRNA decay is important for numerous cellular and developmental processes. Here, we use the patterning gene even-skipped ( eve ) in the early Drosophila embryo to investigate the contribution of mRNA decay to shaping mature expression patterns. Through P-body colocalisation analysis and mathematical modelling of live and fixed imaging data, we present evidence that eve mRNA stability is regulated across stripe 2, with enhanced mRNA decay at the edges of the stripe. To manipulate mRNA stability, we perturbed mRNA decay in the embryo by optogenetic degradation of the 5’ to 3’ exoribonuclease Pacman (Pcm). Depleting Pcm results in larger P-bodies, which accumulate eve mRNAs, and disrupted eve expression patterns. Overall, these data show how eve mRNA instability can function with transcriptional regulation to define sharp expression domain borders. We discuss how spatially regulated mRNA stability may be widely used to sculpt expression patterns during development.
    Date: 2025-02-09
    Authors: Love JC, Beadle LF, Sutcliffe C, Rattray M, Ashe HL.
    Ref: bioRxiv
  22. The ability to quantitatively study mRNA translation using SunTag imaging is transforming our understanding of the translation process. Here, we expand the SunTag method to study new aspects of translation regulation in Drosophila . Repression of the maternal hunchback ( hb ) mRNA in the posterior of the Drosophila embryo is a textbook example of translational control. Using SunTag imaging to quantitate translation of maternal SunTag-hb mRNAs, we show that repression in the posterior is leaky as ∼5% of SunTag-hb mRNAs are translated. In the anterior of the embryo, the maternal and zygotic SunTag-hb mRNAs show similar translation efficiency despite having different UTRs. We demonstrate that the SunTag-hb mRNA can be used as a reporter to study ribosome pausing at single-mRNA resolution, by exploiting the conserved xbp1 mRNA and A60 pausing sequences. Finally, we adapt the detector component of the SunTag system to visualise and quantitate translation of the short gastrulation ( sog ) mRNA, encoding an essential secreted extracellular BMP regulator, at the endoplasmic reticulum in fixed and live embryos. Together, these tools will facilitate the future dissection of translation regulatory mechanisms during development.
    Date: 2025-02-09
    Authors: Pizzey A, Sutcliffe C, Love JC, Akabuogu E, Rattray M, Ashe MP, Ashe HL.
    Ref: bioRxiv
  23. Macrophage phagocytosis is an essential immune response that eliminates pathogens, antibody-opsonized cancer cells and debris. Macrophages can also trogocytose, or nibble, targets. Trogocytosis and phagocytosis are often activated by the same signal, including IgG antibodies. What makes a macrophage trogocytose instead of phagocytose is not clear. Using both CD47 antibodies and a Her2 Chimeric Antigen Receptor (CAR) to induce phagocytosis, we found that macrophages preferentially trogocytose adherent target cells instead of phagocytose in both 2D cell monolayers and 3D cancer spheroid models. Disrupting target cell integrin using an RGD peptide or through CRISPR-Cas9 knockout of the αV integrin subunit in target cells increased macrophage phagocytosis. Conversely, increasing cell adhesion by ectopically expressing E-Cadherin in Raji B cell targets reduced phagocytosis. Finally, we examined phagocytosis of mitotic cells, a naturally occurring example of cells with reduced adhesion. Arresting target cells in mitosis significantly increased phagocytosis. Together, our data show that target cell adhesion limits phagocytosis and promotes trogocytosis.
    Date: 2025-02-08
    Authors: Rollins K, Fiaz S, Morrissey M.
    Ref: bioRxiv
  24. Dosage compensation (DC) in C. elegans utilizes a condensin complex that resembles mitotic condensins, but differs by one subunit, DPY-27. DPY-27 replaces SMC-4, one of the Structural Maintenance of Chromosome (SMC) proteins that is responsible for hydrolyzing ATP, required for condensation of DNA and other mitotic condensin functions. To understand if the ATPase function is required in DC, we first demonstrated that DPY-27 is capable of hydrolyzing ATP in vitro . Then, we used CRISPR/Cas9-mediated genome editing to generate an ATPase mutation in dpy-27 and demonstrated that this mutation results in a loss of DC. Specifically, we found that without ATPase function, DPY-27 containing condensin I DC has reduced capacity to bind DNA, condense the X chromosomes, and facilitate H4K20me1 enrichment on the X-chromosomes. Our results suggest that condensin I DC , like mitotic condensins, uses ATP hydrolysis to perform its functions, making C. elegans DC a model for how activities attributed to mitotic condensins can be used to regulate gene expression.
    Date: 2025-02-08
    Authors: Chawla B, Jatia S, Sloan DE, Csankovszki G.
    Ref: bioRxiv
  25. Fate decisions of T helper (Th) cells are tightly linked to their metabolic states, but precise mechanistic links remain unknown, especially in humans. Using in vitro stimulation in combination with gene editing we studied how metabolic regulation shapes human Th1 cell identity and effector function. Differentiated Th1 cells displayed elevated STAT1 phosphorylation at Tyr701 and Ser727 as well as heightened T-bet and IFNγ expression, which were dampened by CRISPR/Cas9-mediated STAT1 deletion. Metabolic profiling revealed enhanced glycolytic activity in Th1 in comparison to Act.T cells, evidenced by increased extracellular acidification rate, ATP production via glycolysis, glucose uptake, lactate secretion and NADH abundance. SCENITH analysis demonstrated elevated glycolysis-dependent anabolic activity of Th1 cells. Inhibition of glycolysis reduced IFNγ production and STAT1 phosphorylation independent of JAK1/2 activity, STAT1 abundance or SHP-2 activity, implicating glycolysis directly in sustaining STAT1-mediated Th1 functionality. Mechanistically, O-Glycosylation, facilitated by O-Glycosyltransferase, emerged as pivotal in modulating STAT1 activity, as evident through immunoprecipitation and Western blot analysis. Pharmaceutical O-Glycosyltransferase inhibition prevented Th1 differentiation as well as STAT1 O-glycosylation. CRISPR/Cas9 mediated mutation of the O-glycosylation sites Ser499 and Thr510 sites diminished STAT1 Ser727 phosphorylation and IFNγ synthesis. Together, this study highlights glycolysis as key regulator of human Th1 cell identity and effector function, with STAT1 O-Glycosylation selectively maintaining Th1 effector capacity. This mechanism could be explored to safeguard Th1 cells in antiviral immunity and autoimmunity. Graphical abstract
    Date: 2025-02-08
    Authors: Abir AH, Benz J, Frey B, Bruns H, Gaipl US, Schober K, Mougiakakos D, Mielenz D.
    Ref: bioRxiv
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