Results 301-325 of about 1,000
  1. CRISPR-Cas is a defense system of bacteria and archaea against phages. Parts of the foreign DNA, called spacers, are incorporated into the CRISPR array which constitutes the immune memory. The orientation of CRISPR arrays is crucial for analyzing and understanding the functionality of CRISPR systems and their targets. Several methods have been developed to identify the orientation of a CRISPR array. To predict the orientation, different methods use different features such as the repeat sequences between the spacers, the location of the leader sequence, the Cas genes, or PAMs. However, those features are often not sufficient to predict the orientation with certainty, or different methods disagree. Remarkably, almost all CRISPR systems have been found to insert spacers in a polarized manner at the leader end of the array. We introduce CRISPR-evOr , a method that leverages the resulting patterns to predict the acquisition orientation for (a group of) CRISPR arrays by reconstructing and comparing the likelihood of their evolutionary history with respect to both possible acquisition orientations. The new method is independent of Cas type, leader existence and location, and transcription orientation. CRISPR-evOr is thus particularly useful for arrays that other CRISPR orientation tools cannot predict confidently and to verify or resolve conflicting predictions from existing tools. CRISPR-evOr currently confidently predicts the orientation of 28.3% of the arrays in the considered subset of CRISPRCasdb, which other tools like CRISPRDirection and CRISPRstrand cannot reliably orient. As our tool leverages evolutionary information we expect this percentage to grow in the future when more closely related arrays will be available. Additionally, CRISPR-evOr provides confident decisions for rare subtypes of CRISPR arrays, where knowledge about repeats and leaders and their orientation is limited.
    Date: 2025-05-11
    Authors: Fehrenbach A, Mitrofanov A, Alkhnbashi OS, Backofen R, Baumdicker F.
    Ref: bioRxiv
  2. Root cell elongation, the main driver of root growth, is tightly associated with cell wall remodeling, particularly through pectin modifications, which facilitate cell wall loosening and strengthening while maintaining structural integrity. Root cell elongation is precisely regulated by the phytohormone auxin, which has long been known to inhibit this process. The molecular pathways through which auxin influences cell wall modifications remain poorly understood. In this study, we explore the transcriptional regulation of cell wall-related genes by auxin in Arabidopsis thaliana roots. The nuclear auxin pathway altered the expression of numerous cell-wall related genes, suggesting dynamic modification of the cell wall during root cell elongation. We identified novel root-specific polygalacturonases (PGs), enzymes involved in pectin degradation, which we termed POLYGALACTURONASES REGULATED BY AUXIN (PGRAs). PGRAs are expressed specifically in the root epidermis, beginning at the elongation zone. Our results demonstrate that induction of PGRA1 expression initially promotes root cell elongation, while long term overexpression inhibits root growth. Auxin downregulates PGRA1 in the elongation zone, and plants lacking PGRAs fail to increase root growth rate in response to reduced auxin levels. This suggests that auxin downregulates PGRA expression to prevent PGRA-mediated pectin remodeling, thereby contributing to inhibition of root cell elongation. We established a novel link between auxin signaling and pectin modifications in the control of cell growth. These findings provide new insights into the molecular mechanisms through which auxin regulates root cell elongation, highlighting the role of pectin matrix modifications in this process.
    Date: 2025-05-11
    Authors: Kubalová M, Kampová A, Vosolsobě S, Raabe K, Simonaviciene B, Benitez-Alfonso Y, Müller K, Medvecká E, Fendrych M.
    Ref: bioRxiv
  3. ABSTRACT Long non-coding RNAs (lncRNAs) are emerging as key regulatory players of coding gene expression in eukaryotes. Here, we investigate the roles of the lncRNAs SVALKA (SVK) and SVALNA (SVN) in regulating CBF1 and CBF3 gene expression in Arabidopsis under cold stress conditions. We used Native Elongation Transcript Sequencing, CRISPR-Cas9 deletion strategies, and RT-qPCR to analyze the transcriptional dynamics and regulatory mechanisms of SVK and SVN . Our results demonstrate that SVK functions as a cis - and trans -acting lncRNA, regulating both CBF1 and CBF3 through RNAPII collision and chromatin remodeling, while SVN serves a cis role by negatively regulating CBF3 via a RNAPII collision mechanism. We identified isoforms of SVK , originating from distinct transcription start sites and undergo alternative splicing to adapt structural stability, crucial for their regulatory functions. This study elucidates the complex interplay of lncRNAs in gene regulation, highlighting their essential roles in modulating responses to environmental stresses. Our findings contribute to a deeper understanding of the mechanisms underlying lncRNA functionality and their significance in gene regulatory networks in eukaryotes.
    Date: 2025-05-11
    Authors: Rosenkranz I, Mermet S, Zacharaki V, Kindgren P.
    Ref: bioRxiv
  4. Freezing behavior, characterised by attentive immobility as a reaction to a perceived threat, is widely studied in the context of fear, anxiety and stress. To uncover the genetic factors underlying this behavior, we conducted a genome-wide association study (GWAS) in kennel-housed beagle dogs. Our analysis identified a single-nucleotide polymorphism (SNP) in intron 5 of the KCNQ3 gene on chromosome 13 associated with freezing behavior in response to unfamiliar environments and people. To validate this finding, we used a zebrafish model, where CRISPR/Cas9-induced kcnq3 deficiencies led to heightened fear and arousal in two behavioral tests. KCNQ3 is implicated in several neurodevelopmental and psychiatric disorders, and our results highlight its evolutionarily conserved role in modulating fear responses. In dogs, an enriched environment can mitigate the adverse effects of KCNQ3 deficiency by reducing threat perception, highlighting the role of gene-environment interactions in shaping behavioral responses. Teaser KCNQ3 influences fear responses across species, with gene-environment interactions shaping freezing behavior in dogs.
    Date: 2025-05-11
    Authors: Kubinyi E, Sándor S, Tátrai K, Varga Z, Varga ZK, Topál J, Varga M, Jónás D, Turcsán B.
    Ref: bioRxiv
  5. ABSTRACT Temozolomide (TMZ) remains the standard of care for glioblastoma; however, its efficacy is frequently influenced by epigenetic mechanisms, notably the methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter. While MGMT promoter hypermethylation is associated with enhanced responsiveness to TMZ, additional epigenetic determinants of TMZ resistance remain largely undefined. In this study, we established TMZ-resistant glioblastoma cell lines that consistently maintained their resistant phenotype both in vitro and in vivo . Transcriptomic analyses revealed a marked upregulation of MGMT expression in these models. To systematically investigate the epigenetic regulators governing TMZ resistance and cell survival, we conducted CRISPR/Cas9-based functional genomic screens using our focused Epigenetic Knock-Out Library (EPIKOL), which targets 800 chromatin regulators alongside selected positive and negative controls. These unbiased screens validated MGMT as a primary mediator of TMZ resistance, confirming the robustness of our approach. Moreover, dropout screens across multiple resistant cell line models identified Retinoblastoma Binding Protein 4 (RBBP4) as a critical vulnerability. Notably, RBBP4 knockout significantly impaired cell proliferation without affecting MGMT expression, suggesting a distinct mechanism supporting the survival of TMZ-resistant glioblastoma cells. Subsequent transcriptomic profiling following RBBP4 loss demonstrated significant downregulation of cell cycle pathways, particularly the G2/M checkpoint. Live-cell imaging and immunofluorescence analyses further revealed increased cell size and multinucleation in RBBP4-deficient cells, indicative of disrupted mitotic progression. Collectively, our results identify RBBP4 as a key regulator of cell cycle progression and survival in TMZ-resistant glioblastoma and highlight its potential as a novel epigenetic target for therapeutic intervention in recurrent disease.
    Date: 2025-05-10
    Authors: Kala EY, Senbabaoglu Aksu F, Ercan E, Ozbiyik A, Cingöz A, Yedier-Bayram O, Aksu AC, Cavga AD, Yilmaz E, Kok I, Dur Karasayar AH, Kulac I, Syed H, Philpott M, Cribbs AP, Bagci-Onder T.
    Ref: bioRxiv
  6. Abstract Endometrial carcinoma (EC), the most common gynecologic cancer type, encompasses multiple molecular subtypes that have consistent prognostic values and are being adopted in clinical practice to guide treatment decisions. However, it remains unclear whether each of these molecular subtypes have unique therapeutic vulnerabilities that can be exploited for advancing the management of ECs. Through analyzing the genomic features of a panel of 39 EC cell lines, we identified multiple tumor cell lines representing each molecular subtype. Histologic and immunochemical analyses of xenografted tumors from these cell lines confirmed their resemblance of cognate primary EC molecular subtypes, both by histology and the protein expression status of mismatch repair genes, p53 and SWI/SNF members in corresponding subtypes. Further investigation of the publicly available genome-wide CRISPR data for EC cell lines identified multiple specific genetic vulnerabilities in mismatch repair-deficient, p53-abnormal and ARID1A and ARID1B-dual deficient EC cell lines, respectively. Particularly, ARID1A and ARID1B-dual deficient EC cells selectively rely on mitochondria oxidative phosphorylation in vitro and in vivo . Therefore, our study demonstrates the utility of EC cell line models for uncovering and validating therapeutic vulnerabilities of each EC molecular subtype.
    Date: 2025-05-10
    Authors: Li ES, Ho R, Tao R, Choi YW, Shin CY, Chen SY, Gokbayrak B, Senz J, Yao B, Johnston L, Martin SD, Yang E, Carey M, Hennessy BT, Huntsman DG, Geltink RIK, Hoang L, Wang Y.
    Ref: Research Square
  7. Recurrent breast cancer accounts for most disease-associated mortality and can develop decades after primary tumor therapy. Recurrences arise from residual tumor cells (RTCs) that can evade therapy in a dormant state, however the mechanisms are poorly understood. CRISPR-Cas9 screening identified the transcription factors SOX5/6 as functional regulators of tumor recurrence. Loss of SOX5 accelerated recurrence and promoted escape from dormancy. Remarkably, SOX5 drove dormant RTCs to adopt a cartilage-dependent bone development program, termed endochondral ossification, that was confirmed by [ 18 F]NaF-PET imaging and reversed in recurrent tumors escaping dormancy. In patients, osteochondrogenic gene expression in primary breast cancers or residual disease post-neoadjuvant therapy predicted improved recurrence-free survival. These findings suggest that SOX5-dependent mesodermal transdifferentiation constitutes an adaptive mechanism that prevents recurrence by reinforcing tumor cell dormancy.
    Date: 2025-05-10
    Authors: Sreekumar A, Blankemeyer E, Sterner CJ, Pan T, Pant DK, Acolatse S, Turkistani H, Belka GK, Carlin SD, Assenmacher CA, Sellmyer MA, Mankoff DA, Chodosh LA.
    Ref: bioRxiv
  8. The success of cancer immunotherapies is currently limited to a subset of patients, which underscores the urgent need to identify the processes by which tumours evade immunity. Through screening a kinome-wide CRISPR/Cas9 sgRNA library we identified MAP3K7 (TAK1) as a suppressor of CD8+ T cell mediated killing. We demonstrate that TAK1 acts as a cancer-intrinsic checkpoint by integrating signals from T cell-secreted TNF and IFNy effector cytokines to elicit a cytoprotective response. This cytoprotective response profoundly limits the anti-cancer activity these key effector molecules and completely abrogates bystander killing by perforin deficient T cells. Inhibition of the TAK1 checkpoint effectively redirects the combined TNF/IFNy pathway activation to promote inflammatory cell death via RIPK1 and Caspase-8 and simultaneously amplifies the output of the IFNy pathway, thereby priming cells for cytokine-induced cell death. Mechanistically, TAK1 deficiency led to proteasomal degradation of cFLIP, enhancing the formation of Complex II and subversion of other cytoprotective responses. Targeting the TAK1 checkpoint led to profound attenuation of tumour growth in immune competent mice, with minimal impact in immune deficient counterparts. Adoptive cell therapy led to preferential elimination of TAK1 deficient clones. Collectively, our study uncovers a cancer-intrinsic checkpoint controlled by TAK1 activity that switches TNF and IFNy responses from cytoprotective to apoptosis. Cancer cells exploit this to limit cell death in the presence of the cytotoxic lymphoctye cytokines TNF and IFNγ and therapeutic intervention can fully unleash the impact of these effector molecules both on the direct target and bystander cells. These findings highlight the clinical development of TAK1 biologics as a potential strategy to improve cancer immunotherapies through harnessing and enhancing the cytotoxic potential of CTL-derived cytokines.
    Date: 2025-05-10
    Authors: Djajawi TM, Huber A, Mendoza Rivera S, Srivaths A, Salehi M, Gunay G, Gerak C, Neil L, Ozaydin O, Voulgaris O, Al Halawani A, Gannon D, Khoshdoozmasouleh N, Jenkins LJ, Chan KF, Behren A, Ernst M, Mielke LA, Lelliott EJ, Dong H, Feltham R, Sutton VR, Trapani JA, Mariadason JM, Pal B, Martin SJ, Vervoort SJ, Kearney CJ.
    Ref: bioRxiv
  9. ABSTRACT Metastatic breast cancer (MBC) is a life-threatening disease with limited therapeutic options. The immune suppressive tumor microenvironment (TME) limits the potency of the antitumor immune response and facilitates disease progression and metastasis. Our current study demonstrates that p38α is a druggable target in the TME that regulates the outcome of the immune-tumor interaction. The study revealed that systemic blockade of p38α reduces metastasis, and this anti-metastatic response is negated by depletion of CD8 + T cells. Single-cell transcriptomic analysis of the immune-TME showed that pharmacological p38 inhibition (p38i) or tumor-specific inactivation of p38α by CRISPR/Cas9 (p38KO) resulted in a less exhausted and more activated CD8 + T cell phenotype. Immunophenotyping analyses demonstrated that p38 blockade reduced the expression of multiple inhibitory receptors on CD8 + T cells (i.e., PD-1, LAG-3, CTLA-4), indicating a reversal of immune exhaustion and enhanced immune activation systemically and in the TME. In contrast, p38 blockade did not exhibit inhibitory effects on T cells in proliferation assays in vitro and did not affect the proportion of regulatory T cells in vivo . The major negative impact of p38 blockade in vivo was on the myeloid populations, such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). Further, tumor p38α activity was required for the expression of cytokines/chemokines and tumor-derived exosomes with high chemotactic capacity for myeloid cells. Altogether, this study highlights a previously unrecognized p38α-driven pathway that promotes an immune suppressive TME and metastasis, and that therapeutic blockade of p38α has important implications for improving antitumor immunity and patient outcomes. STATEMENT OF SIGNIFICANCE This study highlights a previously unrecognized p38α-driven tumor pathway that promotes an immune suppressive microenvironment and metastasis, and that therapeutic blockade of p38α has important implications for improving antitumor immunity and patient outcomes.
    Date: 2025-05-10
    Authors: Rajan P, Zollo R, Guo Y, Alruwaili M, Zonneville J, Lieberman M, Morreale B, James C, Long M, Olejniczak SH, Barbi J, Abrams SI, Bakin AV.
    Ref: bioRxiv
  10. Chromosomal instability (CIN), a characteristic feature of esophageal adenocarcinoma (EAC), drives tumor aggressiveness and therapy resistance, presenting an intractable problem in cancer treatment. CIN leads to constitutive stimulation of the innate immune cGAS–STING pathway, which has been typically linked to anti-tumor immunity. However, despite the high CIN burden in EAC, the cGAS– STING pathway remains largely intact. To address this paradox, we developed novel esophageal cancer models, including a CIN-isogenic model, discovering myeloid-attracting chemokines – with the chemokine CXCL8 (IL-8) as a prominent hit – as conserved CIN-driven targets in EAC. Using high-resolution multiplexed immunofluorescence microscopy, we quantified the extent of ongoing cGAS-activating CIN in human EAC tumors by measuring cGAS-positive micronuclei in tumor cells, validated by orthogonal whole-genome sequencing-based CIN metrics. By coupling in situ CIN assessment with single-nucleus RNA sequencing and multiplex immunophenotypic profiling, we found tumor cell-intrinsic innate immune activation and intratumoral myeloid cell inflammation as phenotypic consequences of CIN in EAC. Additionally, we identified increased tumor cell-intrinsic CXCL8 expression in CIN high EAC, accounting for the inflammatory tumor microenvironment. Using a novel signature of CIN, termed CIN MN , which captures ongoing CIN-associated gene expression, we confirm poor patient outcomes in CIN high tumors with signs of aberrantly rewired cGAS–STING pathway signaling. Together, our findings help explain the counterintuitive maintenance and expression of cGAS–STING pathway components in aggressive, CIN high tumors and emphasize the need to understand the contribution of CIN to the shaping of a pro-tumor immune landscape. Therapeutic strategies aimed at disrupting the cGAS-driven inflammation axis may be instrumental in improving patient outcomes in this aggressive cancer.
    Date: 2025-05-10
    Authors: Beernaert B, Jady-Clark RL, Shah P, Ramon-Gil E, Lawson NM, Brodtman ZD, Tagore S, Stihler F, Carter AS, Clarke S, Liu T, Zhu W, Erdal E, Easton A, Campo L, Browne M, Ash S, Waddell N, Crosby T, Lord SR, Mann DA, Melero I, de Andrea CE, Tijhuis AE, Foijer F, Hammond EM, Akdemir KC, Leslie J, Izar B, Parkes EE.
    Ref: bioRxiv
  11. A recent study has shown that SKP2 inactivation can prevent cancer initiation by extension of total cell cycle duration without perturbing normal division, which suggests a new strategy for cancer prevention. However, direct in vivo evidence for human SKP2 on cancer initiation and prostatic microenvironment is still lacking and a prostate-specific SKP2 humanized mouse model is critical for developing prostate cancer immunoprevention approaches through targeting human SKP2. We therefore have established a prostate-specific human SKP2 (h SKP2 ) knock-in mouse model by a CRISPR knock-in approach. Overexpression of h SKP2, which is driven by an endogenous mouse probasin promoter, induces prostatic lesions including hyperplasia, mouse prostate intraepithelial neoplasia (mPIN), and low-grade carcinoma and increases prostate weights. Transcriptional profiling by RNA-sequencing analysis revealed significant gene expression alterations in epithelial to mesenchymal transition (EMT), extracellular matrix, and interferon signaling in the prostate of h SKP2 knock-in mice compared to wild-type mice. Single cell deconvolution showed an increase of fibroblasts population and a decrease of CD8 + T cell and B cell populations in the prostate of hSKP2 -knock-in mice. Consistently with these results from the SKP2 humanized mouse, overexpression of hSKP2 in human prostate cancer PC3 cells markedly increased cell migration and invasion and induced the gene expression of EMT and interferon pathways, including FMOD, THY1, PFKP, USP18, IL15, etc. In addition, paired prostate organoids were derived from SKP2 humanized and wild-type mice for drug screening and validated by known SKP2 inhibitors, Flavokawain A and C1. Both of which selectively decrease the viability and alter the morphologies of organoids of h SKP2 knock-in rather than wild-type mice. Our studies provide a well-characterized prostate-specific h SKP2 knock-in mouse model and offer new mechanistic insights for understanding the oncogenic role of SKP2 in shaping the prostatic microenvironment during early carcinogenesis.
    Date: 2025-05-10
    Authors: Song L, Nguyen V, Xu S, Ho KVT, Hoang BH, Yu J, Uchio E, Zi X.
    Ref: bioRxiv
  12. 1 CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems are a fundamental defense mechanism in prokaryotes, where short sequences called spacers are stored in the host genome to recognize and target exogenous genetic elements. Viromics, the study of viral communities in environmental samples, relies heavily on identifying these spacer-target interactions to understand host-virus relationships. However, the choice of sequence search tool to identify putative spacer targets is often overlooked, leading to an unknown impact of downstream inferences in virus-host analysis. Here, we utilize simulated and real datasets to compare popular sequence alignment and search tools, revealing critical differences in their ability to detect multiple matches and handle varying degrees of sequence identity between spacers and potential targets. Finally, we provide general guidelines that may inform future research regarding matching, which is a common practice in studying the complex nature of host-MGE interactions.
    Date: 2025-05-10
    Authors: Neri U, Camargo AP, Bushnell B, Roux S.
    Ref: bioRxiv
  13. ABSTRACT Recent studies have emphasized the significance of biomolecular condensates in modulating gene expression through RNA processing and translational control. However, the functional roles of RNA condensates in cell fate specification remains poorly understood. Here, we profiled the coding and non-coding transcriptome within intact biomolecular condensates, specifically P-bodies, in diverse developmental contexts, spanning multiple vertebrate species. Our analyses revealed the conserved, cell type-specific sequestration of untranslated RNAs encoding key cell fate regulators. Notably, P-body contents did not directly reflect active gene expression profiles for a given cell type, but rather were enriched for translationally repressed transcripts characteristic of the preceding developmental stage. Mechanistically, microRNAs (miRNAs) direct the selective sequestration of RNAs into P-bodies in a context-dependent manner, and perturbing AGO2 or alternative polyadenylation profoundly reshapes P-body RNA content. Building on these mechanistic insights, we demonstrate that modulating P-body assembly or miRNA activity dramatically enhances both activation of a totipotency transcriptional program in naïve pluripotent stem cells as well as the programming of primed human embryonic cells towards the germ cell lineage. Collectively, our findings establish a direct link between biomolecular condensates and cell fate decisions across vertebrate species and provide a novel framework for harnessing condensate biology to expand clinically relevant cell populations.
    Date: 2025-05-10
    Authors: Pessina P, Nevo M, Shi J, Kodali S, Casas E, Cui Y, Richards AL, Park EJ, Chen X, Levin-Ferreyra F, Stevenson E, Krogan NJ, Swaney DL, Ying Q, Chen Q, Brumbaugh J, Di Stefano B.
    Ref: bioRxiv
  14. Prenylnaringenin (PN) compounds, namely 8-prenylnaringenin (8-PN), 3’-prenylnaringenin (3’-PN), and 6-prenylnaringenin (6-PN), are reported to have several interesting bioactivities. This study aimed to validate a biosynthetic pathway for de novo production of PN in Escherichia coli . A previously optimized E. coli chassis capable of efficiently de novo producing naringenin was used to evaluate eleven prenyltransferases (PTs) for the production of PN compounds. As PT reaction requires dimethylallyl pyrophosphate (DMAPP) as extended substrate that has limited availability inside the cells, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 12a (Cas12a) (CRISPR-Cas12a) was used to construct ten boosted DMAPP-E . coli strains. All the PTs, in combination with the naringenin biosynthetic pathway, were tested in these strains. Experiments in 96-well deep well plates identified twelve strains capable of producing PN. E. coli M-PAR-121 with the integration of the 1-deoxy-D-xylulose-5-phosphate synthase (DXS) gene from E. coli ( Ec DXS) into the lacZ locus of the genome ( E. coli M-PAR-121: Ec DXS) expressing the soluble aromatic PT from Streptomyces roseochromogenes (CloQ) and the naringenin biosynthetic pathway was selected as the best producer strain. After optimizing the production media in shake flasks, 160.57 µM of 3’-PN, 4.4 µM of 6-PN, and 2.66 µM of 8-PN were obtained. The production was then evaluated at the bioreactor scale and 397.57 µM of 3’-PN (135.33 mg/L) and 25.61 µM of 6-PN (8.72 mg/L) were obtained. To the best of our knowledge, this work represents the first report of de novo production of PN compounds using E. coli as a chassis.
    Date: 2025-05-10
    Authors: Gomes D, Rodrigues JL, Scrutton NS, Rodrigues LR.
    Ref: bioRxiv
  15. Cas9 provides a powerful tool to interrogate DNA repair and to introduce targeted genetic modifications. However, a major challenge of Cas9-based editing in human embryos is the occurrence of chromosomal abnormalities caused by Cas9 cleavage. Furthermore, mosaicism - different genetic outcomes in different cells, prevent accurate genotyping using a single embryo biopsy. Through timed analysis of editing outcomes during the first cell cycle and timed inhibition of Cas9 using AcrIIA4, we show that most edits occur at least 12 hours post Cas9 injection and therefore after the first S-phase. This timing limits the ability to achieve uniform editing across cells. We found that segmental chromosomal abnormalities and the consequential loss of heterozygosity are common at Cas9 cleavage sites throughout the genome, including at MYBPC3 and at CCR5 loci, for which this has not previously been reported. Surprisingly, inhibiting Cas9 activity 8-12 hours before mitosis does not eliminate chromosomal aneuploidies. This suggests that double-strand break (DSB) repair in human embryos is exceedingly slow, with breaks remaining unrepaired for many hours. Thus, the timing of DSB induction and repair relative to the first S-phase and the first mitosis is intrinsically limiting to preventing mosaicism and maintaining genome stability in embryonic gene editing.
    Date: 2025-05-10
    Authors: Egli D, Marin D, Treff N, Jerabek S, Sung J, Xu J, Talukdar J.
    Ref: bioRxiv
  16. ABSTRACT RNA-guided CRISPR nuclease Cas9 cannot reliably differentiate between single nucleotide variations (SNVs) of targeted DNA sequences determined by their guide RNA (gRNA): they typically exhibit similar nuclease activities at any of those variations, unless the variation occurs with specific sequence contexts known as protospacer adjacent motifs (PAMs). Our approach, “TOP-SECRETS,” generates gRNA variants that allow Cas9 ribonucleoproteins (RNPs) to reliably discriminate between healthy and disease-associated SNVs outside of PAMs.
    Date: 2025-05-10
    Authors: Herring-Nicholas A, Fisher-Huynh S, Josephs EA.
    Ref: bioRxiv
  17. CRISPR-Cas9 guide-RNA design tools can be used to identify guide-RNAs that target single human genomic loci. However, these approaches limit their effect to a single locus. Here, we generate a database of potential individual guide-RNAs that target multiple sites in the genome at once. All guide-RNAs in this database are curated with on- and off-target quantification and enrichment scores for genomic elements such as gene elements, regulatory elements, repetitive elements, and transcription factor motifs. This tool enables rationale mass targeting of genomic loci with a single guide for functional studies. We created a web-app for user-friendly guide-RNA selection ( https://modreklab.shinyapps.io/guiderna/ ).
    Date: 2025-05-10
    Authors: Modrek AS, Huynh K, Do C, Karp J, Ding Y, Zhang Z, Ezhilarasan R, Valor B, Cova G, Skok JA, Sulman EP.
    Ref: bioRxiv
  18. SUMMARY ARID1A is a subunit of the BAF chromatin remodelling complex that is frequently mutated in cancer. It is challenging to predict how ARID1A loss impacts cancer therapy response because it participates in many different cellular pathways. G quadruplex (G4) binding ligands, such as pyridostatin, have shown anticancer effects, but the pathways and genetic determinants involved in the response to G4 ligands are still not fully understood. Here, we show that ARID1A deficient cells are selectively sensitive to pyridostatin when compared with isogenic controls. Sensitivity to pyridostatin was apparent in ovarian and colorectal cancer cell line models, and in vivo studies suggest that G4 ligands hold promise for treating ARID1A deficient cancers. While we find that ARID1A impacts on pyridostatin-induced transcriptional responses, we find that pyridostatin-mediated toxicity in ARID1A-deficient cells is driven by defective DNA repair of topoisomerase-induced breaks. We show that ARID1A-deficient cells are unable to efficiently accumulate non-homologous end joining proteins on chromatin following pyridostatin exposure. These data uncover a role for ARID1A in the cellular response to G4 ligands, and link remodelling to G4 ligand-induced transcriptional and DNA damage responses.
    Date: 2025-05-10
    Authors: Simões-Sousa S, Amin N, Lane KA, Harrod A, Pedersen M, Pardo M, Rasetti-Escargueil C, Kozik Z, Begg KAG, Ribeiro A, Banerjee S, Stucki M, Harrington KJ, Choudhary JS, Downs JA.
    Ref: bioRxiv
  19. Suberin deposition in the root endodermis is critical for plant nutrient acquisition and environmental adaptation. Here, we used an unbiased forward genetic approach based on natural variation across 284 Arabidopsis thaliana accessions to identify novel regulators of suberization. This screen revealed striking diversity in suberin levels and patterns, uncovering broader roles for suberin beyond those observed in the reference accession Col-0. A genome-wide association study pinpointed SUBER GENE1 ( SBG1 ), a previously uncharacterized gene encoding a 129-amino acid protein, as a key regulator of suberin deposition. SBG1 acts through physical interaction with type one protein phosphatases (TOPPs) via conserved SILK and RVxF motifs. Disrupting this interaction abolishes SBG1 function, while topp mutants exhibit enhanced endodermal suberization, mirroring SBG1 overexpression. Our findings uncover a previously unknown regulatory module linking suberin formation to TOPP activity and ABA signaling and provide a framework for improving plant stress resilience through targeted manipulation of root barrier properties.
    Date: 2025-05-10
    Authors: Han J, Lefebvre-Legendre L, Capitão MB, Gully K, Shukla V, Wu Y, Nawrath C, Barberon M.
    Ref: bioRxiv
  20. Cancer therapy has evolved dramatically over the past few decades, progressing from traditional treatments such as surgery, chemotherapy, and radiation therapy to more advanced approaches that target the cellular and molecular mechanisms underlying cancer. Understanding these mechanisms is crucial for developing more effective. While surgery, chemotherapy, and radiation therapy remain the cornerstones of cancer treatment, they are often associated with significant side effects and limited specificity. These treatments work by targeting rapidly dividing cells, but they cannot distinguish between cancerous and normal cells, leading to collateral damage. Cancer is fundamentally a disease of cellular and genetic dysregulation. Understanding the cellular and molecular mechanisms that drive cancer progression is essential for developing targeted therapies that can more precisely attack cancer cells while sparing normal cells. Signal transduction pathways regulate various cellular processes, including growth, differentiation, and survival. In cancer, these pathways are often dysregulated, leading to aberrant cell behavior. For example, the PI3K/AKT/mTOR pathway is frequently activated in cancer, promoting cell growth and survival. Combining different types of therapies can enhance their effectiveness and overcome resistance. For example, combining targeted therapies with immunotherapy or traditional treatments can lead to better outcomes. Researchers are continually exploring new combinations to find the most effective strategies. The field of cancer therapy is rapidly evolving, with ongoing research into new molecular targets, biomarkers for early detection, and strategies to overcome resistance. Advances in technologies such as CRISPR gene editing, artificial intelligence, and personalized medicine are poised to revolutionize cancer treatment. In conclusion, understanding the cellular and molecular mechanisms of cancer is crucial for developing more effective and less toxic therapies. While traditional treatments have their limitations, targeted therapies and new approaches offer hope for better outcomes and improved quality of life for cancer patients. Continued research and innovation are essential to conquer this complex and formidable disease.
    Date: 2025-05-09
    Authors: Rao YT, Rao GH.
    Ref: Preprints.org
  21. Bacteria can encode dozens of different immune systems that protect cells from infection by mobile genetic elements (MGEs). Interestingly, MGEs may also carry immune systems, such as CRISPR-Cas, to target competitor MGEs, but it is unclear when this is favoured by natural selection. Here, we develop and test novel theory to analyse the outcome of competition between plasmids when one carries a CRISPR-Cas system that targets the other plasmid. Our model and experiments reveal that plasmid-borne CRISPR-Cas is beneficial to the plasmid carrying it when the plasmid remains in its host. However, CRISPR-Cas is selected against when the plasmid carrying it transfers horizontally, if the competitor plasmid encodes a toxin-antitoxin (TA) system that elicits post-segregational killing. Consistent with a TA barrier to plasmid-borne CRISPR-Cas, a bioinformatic analysis reveals that naturally occurring CRISPR-Cas-bearing plasmids avoid targeting other plasmids with TA systems. Our work shows how the benefit of plasmid-borne CRISPR-Cas is severely reduced against TA-encoding competitor plasmids, but only when plasmid-borne CRISPR-Cas is horizontally transferred. These findings have key implications for the distribution of prokaryotic defenses and our understanding of their role in competition between MGEs, and the utility of CRISPR-Cas as a tool to remove plasmids from pathogenic bacteria.
    Date: 2025-05-09
    Authors: Sünderhauf D, Ringger JR, Payne LJ, Pinilla-Redondo R, Gaze WH, Brown SP, van Houte S.
    Ref: bioRxiv
  22. Abstract Neurodevelopmental disorders associated with epilepsy are typically linked to postnatal dysfunction of synaptic proteins and ion channels, yet increasing evidence suggests a role for these proteins before birth. The voltage-gated sodium channel Nav1.1, encoded by SCN1A , is well studied postnatally. SCN1A mutations result in a broad range of neurological phenotypes including developmental and epileptic encephalopathies (DEEs including Dravet syndrome, DS) and fetal lethality. Here, we investigated the role of SCN1A dysfunction in early corticogenesis. By integrating data from DS patient-derived forebrain models, a DS mouse model, and DS post-mortem tissue, we report altered G2/M cell cycle transition, with a shift towards earlier neurogenic fate commitment. These changes lead to altered cortical specification at birth and throughout life. The early developmental roles of Nav1.1 complement the well-known postnatal roles in neuronal excitability. These discoveries reveal new insights into the DS pathogenesis and a new non-canonical role for Nav1.1 in early corticogenesis.
    Date: 2025-05-09
    Authors: Zourray C, Mills J, Mills G, Mainardi M, Street J, Bartkiewicz J, Mihaylov S, Sartorelli J, O’Neill N, Carpenter J, Almacellas A, Bagnasco S, Flynn H, Benriassa K, Bari M, Papandreou A, McTague A, Schorge S, Ultanir S, Thom M, Sisodiya S, Kurian M, Colasante G, Barral S, Lignani G.
    Ref: Research Square
  23. Abstract Probiotics ( lactic acid bacteria ) are widely used as microbial feed additives in livestock production and play an important role in preventing and treating animal diarrhea as well as regulating host immune function. In this study, lactic acid bacteria were isolated from the fresh feces of healthy adult female sheep, and their biological characteristics were analyzed. Based on phylogenetic analysis, strain SSF2 was identified as Pediococcus pentosaceus . SSF2 exhibited tolerance to acid, bile salt concentrations, and simulated artificial gastrointestinal environments. The hemolysis test for SSF2 was negative, it was sensitive to commonly used antibiotics, and it demonstrated significant antibacterial and antioxidant activities, indicating its excellent probiotic potential. Whole-genome sequencing (WGS) was performed using the HiSeq 2500 platform and the PacBio system to explore the genetic characteristics of SSF2. The genome was revealed to consist of a circular chromosome and two plasmids, with sizes of 1,785,410 bp, 10,618 bp, and 57,766 bp, and GC contents of 37.23%, 34.95%, and 40.98%, respectively. The genome was predicted to contain five genomic islands, six prophages, and a potential CRISPR gene editing sequence. Functional annotation through databases such as COG, GO, and KEGG revealed that most genes are related to carbon metabolism, protein and amino acid metabolism, nucleotide metabolism, and membrane transport processes. This study indicates that an in-depth understanding of the functionality and genetic characteristics of Pediococcus pentosaceus SSF2 may enable the potential application of this strain in sheep feed supplements.
    Date: 2025-05-09
    Authors: Yao L, Huang Y, Zhang L, Wang Y, Jia J, Wu r, Hai X, Teo SS, Xing Z, Yang Y, Zhang Y, Cheng C, Su S.
    Ref: Research Square
  24. Summary Integrin adhesion complexes mediate cell-extracellular matrix (ECM) interactions and undergo dynamic remodelling to regulate cell adhesion and migration. Here, we demonstrate that tensin 1 (TNS1), a multidomain adhesion adaptor protein linking active integrins with the actin cytoskeleton, undergoes phase separation in cells. Endogenous TNS1 condensates are formed upon focal adhesion disassembly or limited integrin–ECM engagement in both 2D and 3D environments, acting as reservoirs for inactive adhesion proteins. Combining functional experimental approaches with phosphoproteomics, we identify the TNS1 intrinsically disordered region as the main driver of TNS1 condensation and demonstrate a negative regulatory role of phosphorylation on condensate assembly upon activation of stress-responsive kinases. Finally, we confirm the functional effects of phosphorylation-dependent TNS1 condensation on adhesion dynamics and cell migration. Together, our findings highlight TNS1 condensation as a regulatory mechanism controlling local availability of inactive adhesion proteins, with direct implications on adhesion dynamics and cell behaviour.
    Date: 2025-05-09
    Authors: Dibus M, Enkavi G, Chastney M, Vattulainen I, Ivaska J.
    Ref: bioRxiv
  25. METTL9 is an enzyme catalysing N1-methylation of histidine residues (1MH) within eukaryotic proteins. Given its high expression in vertebrate nervous system and its potential association with neurodevelopmental delay, we dissected Mettl9 role during neural development. We generated three distinct mouse embryonic stem cell lines: a complete Mettl9 knock-out (KO), an inducible METTL9 Degron and a line endogenously expressing a catalytically inactive protein, and assessed their ability to undergo neural differentiation. In parallel, we down-regulated mettl9 in Xenopus laevis embryos and characterised their neural development. Our multi-omics data indicate that METTL9 exerts a conserved role in sustaining vertebrate neurogenesis. This is largely independent of its catalytic activity and occurs through modulation of the secretory pathway. METTL9 interacts with key regulators of cellular transport, endocytosis and Golgi integrity; moreover, in Mettl9KO cells Golgi becomes fragmented. Overall, we discovered the first developmental function of Mettl9 and linked it to a 1MH-independent pathway, namely, the maintenance of the secretory system, which is essential throughout neural development.
    Date: 2025-05-09
    Authors: Pandolfini L, Codino A, Spagnoletti L, Olobardi C, Cuomo A, Santos- Rosa H, Palomba M, Margaroli N, GIROTTO S, Scarpelli R, Luan S, Crocco E, Bianchini P, Bannister AJ, Gustincich S, Kouzarides T, Rizzo R, Barbieri I, Cremisi F, Vignali R.
    Ref: bioRxiv
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