Results 1-25 of about 1,000
  1. Abstract 2.1 Background Adipogenesis is a highly organised series of events that facilitates the healthy expansion of adipose tissue, beginning during embryogenesis and continuing throughout life. White adipogenesis protects against lipotoxicity, influencing insulin resistance and obesity-related comorbidities. Brown adipogenesis enhances energy expenditure, thereby counteracting weight gain, lipotoxicity and insulin resistance. Recently, there has been a significant increase in interest regarding adipocyte differentiation, mainly focusing on the interplay between microRNAs (miRNAs) and the transcriptional cascade that governs adipogenesis and metabolic dysfunction. This study aimed to identify miRNAs regulating white and brown adipocyte differentiation and define miRNA action in a stem cell model of adipogenesis. 2.2 Methods Small RNAseq analysis of primary mouse brown and white adipocytes (WAs) identified miR-10b to be upregulated in mature brown adipocytes (BAs). We generated two model systems: 1) immortalized brown pre-adipocytes treated with miRNA inhibitors and 2) CRISPR/Cas9 KO of miR-10b in E14 mouse embryonic stem cells (mESCs). Both cell models were differentiated into mature adipocytes. To unravel the pathways that are affected by miR-10b depletion, a transcriptomic analysis was performed at key time points. 2.3 Results Both cell models showed that miR-10b-5p depletion severely impaired differentiation into mature adipocytes, as indicated by a lack of lipid droplet formation and reduced adipogenic gene expression. Gene expression analysis supports that miR-10b-5p directs embryonic stem (ES) cells towards the mesoderm lineage, promoting commitment to pre-adipocytes by downregulating Gata6 and its downstream target Bmp2. This mechanism appears to be unaffected in BAs. Our study demonstrated that miR-10b-5p regulates the later stages of adipogenesis, at least in part, by downregulating Tub, a direct target of miR-10b-5p. We also confirmed that miR-10b-5p alleviated the halted differentiation phenotypes of adipocytes by supressing the G Protein Signalling pathway mediated by Tubby. 2.4 Conclusions These results evidence that miR-10b inhibition plays a dynamic role in adipocyte biology, as its inhibitory effects manifest differently during the stem cell preadipocyte proliferation state and during the maturation phase of adipocytes. Collectively, our study demonstrated that miR-10b-5p may represent a new potential therapeutic target for lipodystrophy and obesity.
    Date: 2025-08-06
    Authors: Kalenderoglou N, Dimitri F, Gonzalez CN, Vidal-Puig A, Hobbs J, Younis A, Carobbio S, Christian M.
    Ref: Research Square
  2. Abstract Background DOT1L, a histone H3 lysine 79 (H3K79) methyltransferase, is a potential therapeutic target in various malignancies. In the present study, we aimed to clarify the antitumor effect of DOT1L inhibition in breast cancer. Methods Estrogen receptor (ER)-positive/HER2-negative breast cancer cells (MCF7) and ER-negative/HER2-positive cells (SKBR3) were treated with a DOT1L inhibitor (SGC0942, EPZ-5676), after which colony formation assays, cell cycle assays, flow cytometry, gene expression microarray analysis, chromatin immunoprecipitation sequencing (ChIP-seq) and single-cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq) were performed. Genetic ablation of STING was performed using the CRISPR/Cas9 system. Results Treatment with a DOT1L inhibitor suppressed proliferation and induced cell cycle arrest and apoptosis in both ER-positive/HER2-negative and ER-negative/HER2-positive cells. Transcriptome and epigenome analysis revealed that DOT1L inhibition activated transcription of a number of interferon (IFN)-related genes (IRGs) in breast cancer cells. We also found that DOT1L inhibition upregulated type I and type III IFNs and cell surface human leukocyte antigen (HLA) class I expression. Notably, DOT1L inhibition induced DNA damage and upregulated levels of cytoplasmic DNA in breast cancer cells. CRISPR/Cas9-mediated knockout of STING in breast cancer cells significantly suppressed the IFN signaling activated by DOT1L inhibition and attenuated the antitumor effects. Moreover, scATAC-seq analysis revealed that DOT1L inhibition suppressed expression of ERBB2 in HER2-positive breast cancer cells. Conclusions These findings suggest that the anti-breast cancer cell effects of DOT1L inhibition are mediated by multiple mechanisms, including activation of innate immune signaling.
    Date: 2025-08-06
    Authors: Yoshido A, Ishiguro K, Kitajima H, Niinuma T, Kumegawa K, Maezawa M, Tsukahara T, Toyota M, Yorozu A, Sasaki H, Yamamoto E, Kai M, Idogawa M, Torigoe T, Nakase H, Maruyama R, Suzuki H.
    Ref: Research Square
  3. Membrane protection against oxidative insults is achieved by the concerted action of glutathione peroxidase 4 (GPX4) and endogenous lipophilic antioxidants such as ubiquinone and vitamin E. Deficiencies in these protective systems lead to an increased propensity to phospholipid peroxidation and ferroptosis. More recently, ferroptosis suppressor protein 1 (FSP1) was identified as a critical ferroptosis inhibitor acting via regeneration of membrane-embedded antioxidants. Yet, regulators of FSP1 are largely uncharacterised, and their identification is essential for understanding the mechanisms buffering phospholipid peroxidation and ferroptosis. Here, we conducted a focused CRISPR-Cas9 screen to uncover factors influencing FSP1 function, identifying riboflavin (vitamin B2) as a new modulator of ferroptosis sensitivity. We demonstrate that riboflavin, unlike other vitamins that act as radical-trapping antioxidants, supports FSP1 stability and the recycling of lipid-soluble antioxidants, thereby mitigating phospholipid peroxidation. Furthermore, we show that the riboflavin antimetabolite roseoflavin markedly impairs FSP1 function and sensitises cancer cells to ferroptosis. Thus, we uncover a direct and actionable role for riboflavin in maintaining membrane integrity by promoting membrane tolerance to lipid peroxidation. Our findings provide a rational strategy to modulate the FSP1-antioxidant recycling pathway and underscore the therapeutic potential of targeting riboflavin metabolism, with implications for understanding the interaction of nutrients and their contributions to a cell's antioxidant capacity.
    Date: 2025-08-06
    Authors: Skafar V, de Souza I, Ferreira dos Santos A, Porto Freitas F, Chen Z, Donate M, Nepachalovich P, Ghosh B, Tschuck J, Mathur A, Nunes Alves A, Buhr J, Aponte-Santamaria C, Schmitz W, Eilers M, Ubellacker JM, Elling U, Augustin HG, Hadian K, Meierjohann S, Proneth B, Conrad M, Fedorova M, Alborzinia H, Friedmann Angeli JP.
    Ref: bioRxiv
  4. Ferroptosis, a regulated form of cell death driven by excessive lipid peroxidation, has emerged as a promising therapeutic target in cancer. Ferroptosis suppressor protein 1 (FSP1) is a critical regulator of ferroptosis resistance, yet the mechanisms controlling its expression and stability remain mostly unexplored. To uncover regulators of FSP1 abundance, we conducted CRISPR-Cas9 screens utilizing a genome-edited, dual-fluorescent FSP1 reporter cell line, identifying both transcriptional and post-translational mechanisms that determine FSP1 levels. Notably, we identified riboflavin kinase (RFK) and FAD synthase (FLAD1), enzymes which are essential for synthesizing flavin adenine dinucleotide (FAD) from vitamin B2, as key contributors to FSP1 stability. Biochemical and cellular analyses revealed that FAD binding is critical for FSP1 activity. FAD deficiency, and mutations blocking FSP1-FAD binding, triggered FSP1 degradation via a ubiquitin-proteasome pathway that involves the E3 ligase RNF8. Unlike other vitamins that inhibit ferroptosis by scavenging radicals, vitamin B2 supports ferroptosis resistance through FAD cofactor binding, ensuring proper FSP1 stability and function. This study provides a rich resource detailing mechanisms that regulate FSP1 abundance and highlights a novel connection between vitamin B2 metabolism and ferroptosis resistance with implications for therapeutic strategies targeting FSP1 in cancer.
    Date: 2025-08-06
    Authors: Deol KK, Harris CA, Tomlinson SJ, Doubravsky CE, Mathiowetz AJ, Olzmann JA.
    Ref: bioRxiv
  5. Escherichia coli ( E. coli ) is a common bacterium in the human gut and an important cause of intestinal and extraintestinal infections. Some E. coli sequence types (ST) are associated with high pathogenicity. The Extraintestinal Pathogenic E. coli (ExPEC) ST131 is a globally distributed multidrug-resistant human pathogen associated with urinary tract and bloodstream infections. Antibiotic-resistant infections often lead to antibiotic treatment failure, underscoring the need of developing alternative treatments. The highly selective antimicrobial potential of CRISPR-Cas9 has been demonstrated in a range of model organisms. However, the effectiveness of CRISPR-Cas9 in combating ST131-associated infections and the consequences of CRISPR-Cas9 treatment, such as the emergence of escapers, remains unclear. Here, we investigated the antimicrobial activity of CRISPR-Cas9 against ST131 and assessed the frequency and genetic basis of escape. We conjugatively delivered CRISPR-Cas9 to ST131 isolates which carried cefotaxime-resistance-encoding target gene bla CTX-M-15 in the chromosome and characterized escape subpopulations. Two main types of escapers emerged: bla CTX-M-15 -positive escapers carried dysfunctional CRISPR-Cas9 systems and arose at a ~10 -5 frequency. Instead, bla CTX-M-15 -negative escapers presented chromosomal deletions involving bla CTX-M-15 loss. The frequency of bla CTX-M-15 loss depended on the bla CTX-M-15 genetic context. Specifically, bla CTX-M-15 -negative escapers emerged at low frequency (~10 -5 ) in isolates where bla CTX-M-15 was located downstream of insertion sequence (IS) IS Ecp1 , while escapers emerged with high frequency (~10 -3 ) in isolates where bla CTX-M-15 was flanked by IS 26 . This work emphasizes how the genetic context of target genes can drive the outcome of CRISPR-Cas9 tools, where the presence of IS 26 may drive increased frequencies of escape.
    Date: 2025-08-06
    Authors: Morros-Bernaus C, Westley J, Wyrsch ER, Djordjevic SP, Zhang L, Leonard A, Gaze W, Sünderhauf D, van Houte S.
    Ref: bioRxiv
  6. The identification of novel antimalarials with activity against both the liver and blood stages of the parasite lifecycle would have the dual benefit of prophylactic and curative potential. However, one challenge of leveraging chemical hits from phenotypic screens is subsequent target identification. Here, we use in vitro evolution of resistance to investigate nine compounds from the Tres Cantos Antimalarial Set (TCAMS) with dual liver and asexual blood stage activity. We succeeded in eliciting resistance to four compounds, yielding mutations in acetyl CoA synthetase (AcAS), cytoplasmic isoleucine tRNA synthetase (cIRS), and protein kinase G (PKG) respectively. Using a combination of CRISPR editing and in vitro activity assays with recombinant proteins, we validate these as targets for TCMDC-125075 (AcAS), TCMDC-124602 (cIRS), and TCMDC-141334 and TCDMC-140674 (PKG). Notably, for the latter two compounds, we obtained a T618I mutation in the gatekeeper residue of PKG, consistent with direct interaction with the active site, which we modelled with molecular docking. Finally, we performed cross-resistance evaluation of the remaining five resistance-refractory compounds using the Antimalarial Resistome Barcode sequencing assay (AReBar), which examined a pool of 52 barcoded lines with mutations covering >30 common modes of action. None of the five compounds where in vitro evolution of resistance was not successful yielded validated hits using AReBar, indicating they likely act via novel mechanisms and may be candidates for further exploration.
    Date: 2025-08-06
    Authors: Rawat M, Boonyalai N, Smidt C, Luth MR, Chen D, Plater A, Post J, Lin D, McMillan J, Eadsforth T, Moliner-Cubel S, Billker O, Rayner JC, Gamo F, Baragana B, Winzeler E, Lee MC.
    Ref: bioRxiv
  7. Here we present pCASKD, a single-plasmid system for scarless chromosomal editing in Escherichia coli. Our plasmid pCASKD integrates CRISPR-Cas9-mediated counterselection, Lambda-Red recombineering, and temperature-sensitive plasmid curing into a 12 Kb vector to enable kilobase-scale insertions and deletions using a linear dsDNA donor and homologous recombination. Using the flagellar stator motAB locus as a model, we demonstrate that pCASKD enables efficient knock-in and knock-out edits with lower donor DNA input and reduced false positives compared with its parent, multi-plasmid system, No-SCAR. Using a single plasmid reduces transformation steps, accelerates screening, and increases the frequency of correctly edited clones. The protocol can be completed in five days, with potential for further optimization, offering a compact and efficient alternative for microbial genome engineering.
    Date: 2025-08-06
    Authors: Ridone P, Baker MA.
    Ref: bioRxiv
  8. Site-directed RNA editing, especially RNA base editing, allows for specific manipulation of RNA sequences, making it a useful approach for the correction of pathogenic mutations. Correction of RNA transcripts allows therapeutic gene editing in a safe and reversible manner and avoids permanent alterations in the genome. RNA-targeting CRISPR-Cas nucleases (e.g., CRISPR-Cas13) enable delivery within a single adeno-associated virus (AAV) vector for RNA base editing, making the approach clinically feasible. Here, we used the inactive CRISPR-Cas13bt3 (also known as Cas13X.1) fused to the ADAR2 deaminase domain (ADAR2DD) for targeted correction of inherited retinal disease (IRD) mutations. First, we show in vitro that dCas13bt3-ADAR2DD can efficiently correct a pathogenic nonsense mutation (c.130C>T [p.R44X]) found in the mouse Rpe65 gene and recover protein expression in retinal pigment epithelium cells (RPEs). Across clinically reported RPE65 mutations, we observed editing efficiencies ranging from 0% to 60%. In the Rpe65-deficient mouse model of retinal degeneration (rd12), we observed that RNA base editing can recover Rpe65 expression in RPEs and rescue retinal function with no observable adverse effects. We further employed our RNA base editor against the large USH2A gene to assess the promise of RNA base editing for addressing untreatable IRDs caused by genes too large for AAV gene delivery. Against the human USH2A in vitro, we observed up to 60% on-target efficiency. We further found that gRNA mismatches, domain-inlaid ADAR2DD design and nucleocytoplasmic shuttling of the RNA base editor optimised on-target and bystander editing for a highly precise base editor. Against the mouse Ush2a in vitro, we similarly observed up to 60% on-target editing in mammalian cells, while in the Ush2aW3947X mice, we observed ~12% on-target editing, with no impact on retinal structure or function, or transcriptome-wide editing. Overall, our findings demonstrate dCas13bt3-ADAR2DD as a potent tool for gene therapy against IRDs, addressing a significant unmet clinical need in ophthalmology.
    Date: 2025-08-06
    Authors: Kumar S, Chang H, Aubin D, HSIAO Y, Brunet A, Yang J, Huang L, Luu CD, Hewitt AW, Li F, Fry LE, Carvalho LS, Gonzalez Cordero A, Liu G.
    Ref: bioRxiv
  9. Some phages have evolved the ability to cooperate to evade the immunity triggered by their bacterial host. A first exposure to the phage may weaken the host defences and allow later infections to be successful. Because this cooperation requires sequential infections, the phage can invade the host population only if its initial density is sufficiently high in a well-mixed environment. However, most natural bacterial populations are spatially structured. Could spatial structure create more favourable conditions for viral spread? Here we study the effect of spatial structure on the dynamics of cooperative anti-CRISPR (Acr) phages spreading in a population of CRISPR-Cas resistant Pseudomonas aeruginosa bacteria. We show experimentally that spatial structure does not always promote the spread of Acr-phages. In particular, the effect of spatial structure is modulated by the efficacy of the bacterial host’s CRISPR-Cas resistance and by the efficacy of the phage Acr protein. These results are discussed in the light of a mathematical model we developed to describe the spread of the phage. The model allows us to understand the ambivalent effects of spatial structure via its effects on the reproduction and on the persistence of the phage. More generally, we find that spatial structure can have opposite effects on the epidemiological dynamics of the phage, depending on the properties of the Acr protein encoded by the phage. This joint experimental and theoretical work yields a deeper understanding of the spatial dynamics of cooperative strategies in phages.
    Date: 2025-08-06
    Authors: Chevallereau A, Kumata R, Fradet O, Lion S, Westra ER, Sasaki A, Gandon S.
    Ref: bioRxiv
  10. Understanding the gene regulatory mechanisms underlying brain function is crucial for advancing knowledge of the genetic basis of neurologic diseases. Cis-regulatory elements (CREs) play a pivotal role in gene regulation, and their evolutionary conservation can offer valuable insights. Importantly, the function and evolution of CREs are affected not only by primary sequence, but also by the cis- and trans-regulatory context. However, comparative functional analyses across species have been limited, leaving how these regulatory landscapes evolve in the brain largely unresolved. Here, we generated single-nucleus multiomic (snRNA- and snATAC-seq) data from cortex tissue across nine mammalian species and identified candidate CREs (cCREs) in a cell type-specific manner. We developed a multidimensional framework of conservation to assess sites of shared function that integrates sequence, chromatin accessibility, and enhancer-gene associations. Using massively parallel reporter assays (MPRA) in human neural progenitor cells and neurons, we measured activity of cCREs including both conserved and human-specific regions. CRISPR interference validated conserved enhancer function, including at neurodevelopmentally important genes like FAM181B. Motif enrichment identified transcription factors distinguishing conserved versus recent cCREs. Linkage disequilibrium score regression indicated that both conserved and human-specific cCREs were enriched for neuropsychiatric GWAS risk, while neurodegenerative risk was confined to conserved elements. Our findings define functional dimensions of enhancer conservation and demonstrate how regulatory evolution shapes human brain biology and disease susceptibility.
    Date: 2025-08-06
    Authors: Anderson AG, Rogers BB, Barinaga EA, Loupe J, WaMaina E, Johnston SQ, Limbo HL, Gardner EA, Moyer AJ, Gross AL, Martin DR, Thyme SB, Rizzardi LF, Myers RM, Cooper GM, Cochran JN.
    Ref: bioRxiv
  11. Genetic interactions are typically studied by looking at the phenotype that results from disruption of pairs of genes, as well as from higher order combinations of perturbations. Systematically interrogating all pairwise combinations provides insights into how genes are organized into pathways and complexes to sustain cellular homeostasis and how interacting genes respond to stressors and external signals. Genetic interactions have been studied extensively in yeast, due, in part, to the availability of a systematic collection of gene knockouts, and the development of Synthetic Genetic Array (SGA) technology. In contrast, such approaches are more challenging in human cells and therefore comparable data for human cells is scarce. This study introduces an innovative approach to functionally characterize genetic interactions in human cells through CRISPR/Cas9 screens using a pooled genome-wide knockout library in NALM6 cells. By combining a single guide RNA (sgRNA) targeting the gene of interest (aka the query) in cells already infected with an inducible genome-wide sgRNA pool, it is possible to achieve near saturation of genome-wide double knockouts. We conducted 26 of these screens, which we term "gene by genome-wide" knockout screens. This approach can be rapidly performed, in part, because it bypasses the need to generate genotyped isogenic knockout clones. Data from these screens identified both expected and novel synthetic lethal and synthetic rescue interactions, demonstrating that this strategy is effective for large-scale genetic research in human cells. Additionally, we show that these GBGW screens can be combined with chemical perturbation to reveal new synthetic interactions that are not apparent without drug treatment. Finally, we show that cDNA overexpression can be incorporated with genome-wide knockouts to systematically explore gain-of-function scenarios. The complete dataset is accessible on the ChemoGenix website (URL: https://chemogenix.iric.ca)
    Date: 2025-08-06
    Authors: Chatr-aryamontri A, Zhang L, Thiombane NK, Archambault V, Carreno S, Di Noia JM, Toposiviric I, Lessard J, Roux PP, Pilon N, Schimmer A, Tyers M, Nislow C, Mader S, Wilhelm B, Bertomeu T.
    Ref: bioRxiv
  12. Human papillomavirus (HPV) is a major etiological factor in cervical, anal, and oro-pharyngeal cancers. Although prophylactic vaccines have substantially reduced infec-tion rates, effective therapeutic options for established HPV-associated malignancies remain limited. This review provides an up-to-date overview of emerging strategies to treat HPV-driven tumors. Key approaches include immune checkpoint inhibitors, therapeutic vaccines such as VGX‑3100 and PRGN‑2012, and gene-editing tools like CRISPR/Cas9. Epigenetic drugs, particularly histone deacetylase inhibitors, show promise in reactivating silenced tumor suppressor genes and enhancing antitumor immunity. In addition, natural bioactive compounds and plant-derived molecules are being explored as complementary anti-HPV agents, while drug repurposing and combination therapies offer cost-effective opportunities to broaden treatment options. We also highlight the role of patient-derived organoid models as powerful platforms for personalized drug screening and functional assessment. By integrating these therapeutic innovations with precision oncology approaches, this review outlines a multi-dimensional framework aimed at improving clinical outcomes and quality of life for patients with HPV-associated cancers.
    Date: 2025-08-05
    Authors: Cakir MO, Kayhan G, Yilmaz B, Ozdogan M, Ashrafi GH.
    Ref: Preprints.org
  13. Terpenoids, vital pharmaceutical compounds, face production challenges due to low yields in native plants and ecological concerns. This review synthesizes recent advances in metabolic engineering strategies implemented across three complementary platforms: native medicinal plants, microbial systems, and heterologous plant hosts. We elucidate how the "Genomic Insights to Biotechnological Applications" paradigm, empowered by multi-omics technologies such as genomics, transcriptomics, metabolomics, etc., drives research advancements. These technologies facilitate the identification of key biosynthetic genes and regulatory networks. CRISPR-based tools, enzyme engineering, and subcellular targeting are highlighted as transformative strategies. Significant yield improvements have been demonstrated, with artemisinin and paclitaxel precursors showing considerable increases in production through strategic co-expression and optimization techniques. Persistent challenges such as metabolic flux balancing, cytotoxicity, and scale-up economics are discussed alongside emerging solutions including machine learning and photoautotrophic chassis. We conclude by outlining a roadmap for industrial translation that emphasizes the critical integration of systems biology and synthetic biology approaches to accelerate the transition of terpenoid biomanufacturing from discovery to commercial scale.
    Date: 2025-08-05
    Authors: Guo C, Xu S, Guo X.
    Ref: Preprints.org
  14. Genome editing technologies including CRISPR/Cas9, TALENs, and ZFNs offer a unique opportunity to eradicate HPV by directly disrupting its oncogenes E6 and E7, thereby restoring tumour‑suppressor pathways. In this review, we provide a comprehensive overview of recent advances in HPV‑targeted genome editing, summarising key preclinical findings that demonstrate tumour regression and apoptosis in HPV‑positive models, as well as the first‑in‑human clinical trials assessing safety and feasibility of local CRISPR‑based therapies. We also compare the relative strengths and limitations of each editing platform, discuss delivery strategies, and highlight their potential integration with immunotherapy and standard cancer treatments. While genome editing shows unprecedented precision and durability in targeting viral oncogenes, challenges such as efficient delivery, minimising off‑target effects, and navigating regulatory and ethical considerations remain. Continued optimisation of high‑fidelity nucleases, tissue‑specific delivery vehicles, and personalised guide design will be essential to translate these promising approaches into routine oncology practice. Genome editing thus represents a paradigm shift in HPV therapy, with the potential to transform management of both persistent infections and established cancers.
    Date: 2025-08-05
    Authors: Cakir MO, Selek M, Yilmaz B, Ozdogan M, Ashrafi GH.
    Ref: Preprints.org
  15. DNASE1L3 is a key endonuclease, essential for proper fragmentation and clearance of cell-free DNA (cfDNA). The p.R206C common variant impairs DNASE1L3 secretion and activity, causing aberrant cfDNA fragmentation and therefore affecting liquid biopsy-based screening and diagnostics. Existing studies on DNASE1L3 relied on resource-intensive murine models or plasmid-based overexpression, which do not accurately represent native expression. To address this, we developed an isogenic HEK293T cell line model by using CRISPR Prime Editing for endogenous expression of DNASE1L3 R206C . We analyzed the cfDNA composition directly from conditioned culture medium and found that fragment size distributions in mutant cells mimics the hypofragmented profiles previously observed in plasma samples from p.R206C carriers. We also showed that in vitro treatment of hypofragmented cfDNA with recombinant wildtype DNASE1L3 could enrich for mononucleosomal fragments, with fragment end-motifs characteristic of DNASE1L3 cleavage activity. This could open avenues for DNASE1L3 as a candidate pre-treatment agent to improve the accuracy and efficiency of cfDNA sequencing-based diagnostics in hypofragmented liquid biopsies. These findings demonstrate that our isogenic cell line model provides a controlled system to study cfDNA fragmentation biology and DNASE1L3 function.
    Date: 2025-08-05
    Authors: Kohabir KA, Balk JA, Nooi LO, Papaioannou D, Wolthuis RM, Sistermans EA, Linthorst J.
    Ref: bioRxiv
  16. CRISPR-Cas9 has revolutionized plant genome editing by enabling precise introduction of insertion/deletion (indel) mutations, critical for functional genomics and crop improvement studies. Sanger sequencing, combined with bioinformatics tools like the INDIGO webserver from Gear Genomics, is essential for validating these mutations. However, manual analysis of large numbers of Sanger sequencing (.ab1) files is labor-intensive, particularly when analyzing multiple guide RNA (gRNA) target regions. We developed a Python-based automation pipeline using Selenium with integrated highlighting of protospacer adjacent motif (PAM) regions in the resulting HTML reports. This pipeline enhances scalability of Sanger sequence data analysis and improves result interpretability by automating PAM region identification and supporting multiple gRNA regions. This tool significantly accelerates CRISPR-Cas9-mediated mutation analysis, offering a high throughput, reproducible solution for genome editing research.
    Date: 2025-08-05
    Authors: Suresh V, Girish C, Tavva VSS.
    Ref: bioRxiv
  17. Efficient utilization of complex biomass-derived sugars and tolerance to inhibitors are key requirements for the viability of lignocellulosic-based biorefineries. In this study, a two-stage evolution of an industrial yeast strain engineered with a xylose isomerase pathway yielded strain AceY.14, which exhibited improved fermentative performance and increased tolerance to acetic acid. Whole-genome sequencing of the evolved strain identified SNPs in ZWF1, a component of the pentose phosphate pathway (PPP), and in the G1 cyclin gene CLN3, both of which were functionally validated through CRISPR and reverse engineering. The zwf1E191D mutation reduced xylitol accumulation, alleviating inhibition of xylose isomerase and enhancing flux through the non-oxidative branch of the PPP, while the frameshift cln3T556fs mutation unexpectedly improved acetic acid tolerance and xylose consumption in the evolved strain, also affecting cell size and growth. Genome sequencing of AceY.14 also revealed a significant reduction in the xylA gene copy number, likely decreasing the metabolic burden associated with high xylose isomerase expression. A synergistic effect was observed in the double mutant lacking both ISU1 and ZWF1, further boosting xylose consumption rates. A diploid derivative (AceY-2n) demonstrated high productivity and robustness in fermentations using hydrolysates from various lignocellulosic feedstocks, highlighting the strain potential for industrial-scale applications. These findings reveal novel metabolic targets for strain optimization and offer valuable insights for the rational engineering of yeast platforms for sustainable biofuel and bioproduct production.
    Date: 2025-08-04
    Authors: Santos LVd, Palermo GCdL, Costa PEdS, Almeida LD, Carazzolle MF, Pereira GAG.
    Ref: bioRxiv
  18. ABSTRACT KRAS mutations are among the most prevalent oncogenic drivers in non-small cell lung cancer (NSCLC), yet the mechanisms of therapeutic resistance to KRAS inhibitors in these cancers remains poorly understood. Here, we deploy high-throughput CRISPR base editing screens to systematically map resistance mutations to three mechanistically distinct KRAS-targeted therapies, including KRAS-G12C(OFF) inhibitor (adagrasib), RAS(ON) G12C-selective tri-complex inhibitor (RMC-4998), and RAS(ON) multi-selective tri-complex inhibitor (RMC-7977). Using both a saturation Kras tiling approach and cancer-associated mutation library, we identify common and compound-selective second-site resistance mutations in Kras , as well as gain-of-function and loss-of-function variants across cancer-associated genes that rewire signaling networks in a context-dependent manner. Notably, we identify a recurrent missense mutation in capicua ( Cic ), that promotes resistance to RMC-7977 in vitro and in vivo. Moreover, we show that targeting NFκB signaling in CIC-mutant cells can resensitize them to RAS pathway inhibition and overcome resistance.
    Date: 2025-08-04
    Authors: Diaz BJ, Kops M, Bernardo S, Schmidt H, Grankowsky E, Vega A, Zhang C, Bott M, Skamagki M, Tomlinson A, Vita NA, Katti A, Labrecque MP, Aronchik I, Singh M, Dow LE.
    Ref: bioRxiv
  19. Background/Objectives Hepatoblastoma (HB) is the most common form of pediatric liver cancer, with the vast majority of these tumors evidence of mutation and/or deregulation of the oncogenic transcription factors β-catenin (B), YAP (Y) and NRF2 (N). HB research has been hampered by a paucity of established cell lines, particularly those bearing these molecular drivers. All combinations of B, Y and N (i.e. BY, BN, YN and BYN) are tumorigenic when over-expressed in murine livers but it has not been possible to establish cell lines from primary tumors. Recently, we found that concurrent Crispr-mediate targeting of the Cdkn2a tumor suppressor locus allows for such immor-talized cell lines to be generated with high fidelity. Methods We generated 5 immortalized cell lines from primary Cdkn2a -targeted BN and YN HBs and characterized their properties. Notably, 4 of the 5 retain their ability to grow as subcutaneous or pulmonary tumors in the immune-competent mice from which they originated. Most notably, when maintained under hypoxia conditions for as little as 2 days, BN cells reversibly up-regulated the expression of numerous endothelial cell (EC)-specific genes and ac-quired EC-like properties that benefited tumor growth. Conclusions The above approach is currently the only means by which HB cell lines with pre-selected, clinically relevant oncogenic drivers can be generated and the only ones that can be studied in immune-competent mice. Its generic nature should allow HB cell lines with other oncogenic drivers to be derived. A collection of such cell lines will be useful for studying tumor cell-EC trans-differentiation, interactions with the immune environment and drug sensitivities. Simple Summary Most hepatoblastomas (HB) are associated with aberrant expression of β-catenin (B), YAP (Y) and/or NRF2 (N) transcription factors and can be modeled in mice by over-expressing pairwise of triple combination of these. Virtually no human or murine HB cell lines exist that bear these mutations. We describe here an efficient way to generate cell lines from primary BN and YN tumors. Moreover, one of the BN lines shows a remarkable ability to trans-differentiate into endothelial cells under hypoxic conditions that may facilitate angiogenesis. These cell lines along with previousl-derived BN and BYN lines showed similar sensitivities to drugs commonly used to treat HB. Because the approach for cell line derivation we describe is quite general, it should allow for the generation of additional lines driven by less common factors. A collection of such permanent and well-characterized cell lines will facilitate studies that are difficult or impractical to perform in vivo .
    Date: 2025-08-03
    Authors: Chen K, Toksoz A, Henchy C, Knapp J, Lu J, Ranganathan S, Wang H, Prochownik EV.
    Ref: bioRxiv
  20. Type 1 diabetes can be cured by β–cell replacement in principle, yet recurrent autoimmunity and transplantation barriers rapidly destroy implanted cells. Genome–wide CRISPR screening by Cai et al . highlighted RNLS and HIVEP2 as candidate genes, but their value outside an autoimmune setting is unknown. Here, it was evaluated whether single-gene knockout of RNLS or HIVEP2 could similarly protect β-cell grafts against allo- and xenogeneic rejection. Murine β–TC–6 and human EndoC–βH1 cell lines were genetically edited using CRISPR-Cas9 to knockout RNLS or HIVEP2, and editing efficiencies were confirmed via T7 endonuclease I assay and TIDE analysis. Functional characterization indicated that RNLS deletion modestly impaired glucose-stimulated insulin secretion in murine cells, whereas HIVEP2 deletion showed no functional alterations in either cell line. For in vivo assessment, genetically edited β-cell spheroids were subcutaneously transplanted into CD-1 mice to model allo- (murine β-cells) and xenogeneic (human β-cells) rejection scenarios. Bioluminescence imaging revealed no protective effects of RNLS or HIVEP2 deletion, with grafts from both knockout groups displaying identical rejection kinetics compared to controls. These findings indicate that single-gene deletions of RNLS or HIVEP2 are insufficient for conferring meaningful protection against allo- or xenorejection, highlighting the necessity for combinatorial genome editing strategies or complementary biomaterial-based immunomodulation to achieve effective and sustained β-cell graft survival.
    Date: 2025-08-02
    Authors: Karaoglu IC, Odabas A, Onder T, Kizilel S.
    Ref: bioRxiv
  21. Anoikis is an apoptotic cell death program triggered upon detachment from surrounding extracellular structures. However, the ability to evade cell death by anoikis in the presence of apoptosis-inducing stimuli is necessary for the formation of malignant tumors and progression to metastasis. Our findings indicate that the BRN2 (POU3F2) transcription factor is associated with anoikis resistance in melanoma cells. However, the BRN2 signaling cascade driving anoikis resistance remains unknown. Herein, we employed genome-wide CRISPR screens to validate BRN2 as a driver of anoikis resistance. Small molecule inhibition of BRN2 in melanoma cell lines with acquired anoikis resistance resensitized to death by anoikis in ultra-low attachment conditions. Our quantitative mass spectrometry analysis revealed that BRN2 functionally impacts oxidative phosphorylation and mitochondrial activity, whereby probes designed to inhibit BRN2 induced apoptosis and mitochondrial fragmentation through the MAPK and NF-κB signaling pathways and reduction in PPARɣ expression. Our study suggests that inhibition of BRN2 might allow the targeting of metastatic cells in circulation, and sensitizes cells to BRAF-targeted therapy, improving the prognosis for melanoma patients. Abstract Figure Graphical Abstract. Role of BRN2 in driving anoikis resistance in melanoma. Upon detachment from the extra-cellular matrix (ECM) melanoma cells must evade cell death by anoikis to seed distant metastases. This study expanded the understanding of the role of the BRN2 transcription factor as a driver of resistance to anoikis in melanoma. The use of small molecule inhibitors targeting BRN2 revealed that the transcription factor drives anoikis resistance via the MAPK and NF-κB signaling pathways, resulting in PPARγ dysregulation and subsequently driving mitochondrial dysfunction. Green boxes = previously published drivers of anoikis resistance in melanoma. Blue box = changes to mitochondrial function following inhibition of BRN2 as determined by proteomics analysis.
    Date: 2025-08-02
    Authors: Neuendorf HM, He X, Adams MN, Tran KA, Smith AG, Bernhardt PV, Williams CM, Simmons JL, Boyle GM.
    Ref: bioRxiv
  22. CRISPR/Cas9-based homing gene-drives (homing-drives) hold enormous potential as control tools for mosquito disease-vectors. These genomically-encoded technologies spread themselves through target populations by creating double-stranded DNA breaks on homologous chromosomes, into which the homing-drives are copied (‘homed’). Homing is dependent on sequence homology between the genomic regions flanking the transgene insertion and the break site. Homing efficiency (i.e. copying rate) substantially impacts the power of these systems: less efficient homing-drives spread slower, have fewer applications and are more resistance-prone. Understanding what influences homing-drive efficiency is therefore vital to the successful use of these technologies. Here we report a novel mechanism by which a homing-drive’s efficiency can be significantly impaired by natural sequence variation within a population into which it is spreading. Using a kmo -targeting ‘split’ homing-drive in the West Nile virus mosquito Culex quinquefasciatus , we found that target-site heterology (sequence mismatch between the genomic regions flanking the target cut-site and the homing-drive transgene) of less than 10% reduced homing efficiency by up to 54%. While substantial research effort has been dedicated to increasing homing-drive efficiency through optimisation of within-construct components, our results highlight that the real-world efficacy of these systems may in part depend on variation beyond these controllable factors.
    Date: 2025-08-01
    Authors: Harvey-Samuel T, Kaur R, Leftwich P, Feng X, Gantz V, Alphey L.
    Ref: bioRxiv
  23. Abstract Tn7 mobile genetic elements are known for their sophisticated target-site selection mechanisms and, in some cases, programmability. Recognition of target sites is mediated by designated transposon-encoded proteins and modulated by host factor proteins. In the case of the CRISPR-associated Tn7 elements from the type V-K, the ribosomal protein uS15 is an integral component of recruitment complex that promotes R-loop completion. Previous biochemical work also revealed that the ribosomal protein uL29 and the acyl carrier protein (ACP) influence Tn7 transposition frequency in vitro . However, how uL29 and ACP regulate the formation of the Tn7 targeting complex remains unclear. The prototypical Tn7 element encodes a heteromeric transposase (TnsAB), a AAA+ adaptor (TnsC), and two target-site selection proteins (TnsD and TnsE). TnsD targets a highly conserved site at the end of the glmS gene ( attTn7 ). However, poor protein stability has precluded the molecular characterization of how TnsD recognizes its target site. Here, we show that ACP and uL29 interact with the C-terminal region of TnsD through reciprocal electrostatic interactions, in turn, mitigating its tendency to aggregate. Additionally, we identify the uL29 and ACP residues that mediate the interaction with TnsD and stimulate DNA binding. These results unveil unique features of the TnsD-mediated target-site selection complex.
    Date: 2025-08-01
    Authors: Camacho SBL, Matthews LA, Guarné A.
    Ref: Research Square
  24. CRISPR/Cas9-based genome editing in the model bryophyte Physcomitrium patens (commonly known as Physcomitrella) is widely used for gene knockout via small insertions or deletions (indels). However, this approach may leave residual gene activity and typically requires sequencing-based validation. In this study, we established an efficient strategy for generating large, targeted deletions across multiple genes using dual-gRNA targeting. We first compared the efficiency of polycistronic tRNA-gRNA arrays to conventional gRNA constructs expressed under individual promoters, using the checkpoint protein gene MAD2 as a target. We found that a polycistronic construct doubled the frequency of large gene deletions compared to a conventional design. We then demonstrated that simultaneous deletion of two or four genes, targeting the katanin and TPX2 gene families, respectively, can be achieved in a single transformation event. The polycistronic system also increased deletion frequencies in the multiplex context, with up to 42% efficiency for individual genes and successful recovery of quadruple mutants. As a drawback, we confirmed that deletion efficiency varied substantially among individual gRNA pairs, indicating that gRNA design remains a critical factor in multiplex editing. This study establishes a versatile and scalable framework for generating multi-gene deletion mutants in P. patens , facilitating functional genomics and biotechnological applications requiring precise gene removal.
    Date: 2025-08-01
    Authors: Kozgunova E.
    Ref: bioRxiv
  25. In contrast to animals, plants have a high regenerative capacity, and they can form new organs and even complete individuals from a few cells present in adult tissues, either in response to injury or to the alteration of their environment. In this study, we describe the isolation and characterization of the more adventitious roots1-1 ( mars1-1 ) mutant, which exhibits enhanced regenerative potential upon wounding in tomato hypocotyl explants. Additionally, the mars1-1 fruits exhibited a rough surface due to the ectopic proliferation of subepidermal cells, which formed callus-like structures on the cuticle. The MARS1/ROUGH gene encodes a conserved lysine-specific histone demethylase, SlLSD1, which regulates a variety of processes in metazoans, including cell proliferation, stem cell pluripotency, and embryogenesis. Two CRISPR/Cas9 null alleles, mars1-2 and mars1-3 , were generated and their pleiotropic phenotype was characterized. We found elevated levels of H3K4me1 in mars1/rough seedlings, which suggests that SlLSD1 is required for the demethylation of this histone mark. To ascertain the impact of altered epigenetic marks in the mars1/rough mutants on gene expression regulation, we conducted a transcriptome analysis using a variety of RNA-Seq studies on tomato hypocotyls. By employing specific bioinformatic workflows and leveraging on the resolution of directional RNA-Seq data, we have identified over several dozen distinct genomic regions that exhibit de novo expression in the mars1/rough mutants. One such region includes a novel B-type cyclin gene, which is upregulated in the mars1/rough mutants and may account for the observed phenotypes. Our findings indicate that SlLSD1 plays a role in the establishment and maintenance of silencing in specific genomic regions that are essential for tissue-specific reprogramming.
    Date: 2025-08-01
    Authors: Larriba E, Bres C, Alaguero-Cordovilla A, Petit J, Riyazuddin R, Mauxion JP, Caballero L, Bakan B, Esteve-Bruna D, Benhamed M, Rothan C, Pérez-Pérez JM.
    Ref: bioRxiv
first · previous · 1 · 2 · 3 · 4 · 5 · 6 · 7 · 8 · 9 · 10 · next · last