Results 926-950 of about 1,000
  1. Colonisation of the mosquito by the malarial parasite is critically reliant on the invasive ookinete stage. Ookinete invasion of mosquito is coordinated by the apical complex, a specialised parasite structure containing components for secretion, attachment and penetration. While studies have investigated cytoskeletal and secretory elements, it is currently unknown if signalling modules are present or functional at the apical complex. Here we elucidate the role of a cryptic cyclic nucleotide binding protein which we name CBP-O. PbCBP-O showed a marked localisation to the ookinete apex and disruption of the protein severely compromised ookinete invasion of mosquitos. Domain dissection analysis revealed that the N- and C-termini have distinct functions. Intriguingly, PbCBP-O exhibits dual binding specificity to both cGMP and cAMP. Our findings suggest the apical tip of the ookinete is a platform to transduce cyclic nucleotide signals essential for malaria parasite transmission. Author summary Malaria parasites complete their life-cycle within mammalian and mosquito hosts requiring specialised forms to invade and establish infection in host tissue. In order to transmit to the mosquito, the parasite forms a motile ookinete that uses the apical end to attach and penetrate the mosquito midgut membrane. Previous studies have identified several proteins localised to the apex of the ookinete. However, functions of many of these proteins are unknown. Here, we dissect the role of a putative cyclic nucleotide binding protein, CBP-O, in establishing ookinete infection within the mosquito. We discovered that in the absence of the protein, efficiency of transmission is significantly compromised. Interestingly the ability of ookinetes to move was not affected, but the parasites were unable to pass through the midgut membrane. We also reveal that both N- and C-terminal domains of the protein perform distinct functions and are both required for parasite transmission. Sequence and spatial conservation of the protein across the phylum Apicomplexa suggests an equivalent and crucial function of CBP-O in these parasites.
    Date: 2025-01-30
    Authors: Kwecka D, Eigminas E, Liu L, Kim C, Philip N.
    Ref: bioRxiv
  2. CRISPR gene editing is a transformative technology for addressing genetic diseases, but delivery constraints have largely limited its therapeutic applications to liver-targeted and ex vivo therapies. Here, we present the discovery and engineering of NanoCas, an ultracompact CRISPR nuclease capable of extending CRISPR’s reach in vivo beyond liver targets. We experimentally screened 176 ultracompact CRISPR systems found in metagenomic data and applied protein engineering approaches to enhance the editing efficiency of NanoCas. The optimized NanoCas exhibits potent editing capabilities across various cell systems and tissues in vivo when administered via adeno-associated viral (AAV) vectors. This is accomplished despite NanoCas being approximately one-third the size of conventional CRISPR nucleases. In proof-of-concept experiments, we observed robust editing with our optimized NanoCas in mouse models targeting Pcsk9, a gene involved in cholesterol regulation, and targeting exon splice sites in dystrophin to address Duchenne muscular dystrophy (DMD) mutations. We further tested the efficacy of our NanoCas system in vivo in non-human primates (NHPs) resulting in editing levels above 30% in muscle tissues. The compact size of NanoCas, in combination with robust nuclease editing, opens the door for single-AAV editing of non-liver tissues in vivo , including the use of newer editing modalities such as reverse transcriptase (RT) editing, base editing, and epigenetic editing.
    Date: 2025-01-30
    Authors: Rauch BJ, DeLoughery A, Sper R, Chen S, Yunanda S, Masnaghetti M, Chai N, Lin JC, Neckelmann A, Bjornson Y, Espino DP, Sancio A, Schmitt C, Scholes C, Shah R, Narendra PK, Ansaloni S, Tan S, Rukmini SJ, Somaiah S, Suresh S, Minami S, Tymoshenko S, Wright W, Xu S, Broughton J, Retwitzer MD, Bobbin M, Yuan D, Abe K, DeWitt M, Zhang B, Harrington LB.
    Ref: bioRxiv
  3. Abstract Wheat is the staple food for 40% of the world, providing 20% of dietary energy and protein. However, along with providing nutrition, wheat contains several anti-nutritional macromolecules. Amylase/Trypsin inhibitors (ATIs) are one such macromolecular proteins which have been known to cause allergic reactions like baker's asthma, auto-immunogenic reactions like Non-Celiac Wheat Sensitivity, and primary triggers for Celiac Disease in some predisposed humans. Bread wheat varieties without ATI molecules or with reduced activity have not yet been developed. Here, multiple genes of major ATI protein molecules were mutated using tRNA-based multiplex CRISPR/Cas9 genome editing technology. ATI proteins were extracted from wheat flours of gene-edited wheat lines along with unedited plants and subjected to quantification, detection by SDS-PAGE, fractionation by HPLC, and assayed the α-amylase and trypsin inhibition activity. Gene-edited Bobwhite wheat plant produced seeds with reduced (up to 30.61%) ATI content, which resulted in a reduction in α-amylase and trypsin inhibition activity to 50.74% and 44.90%, respectively. Another variety of bread wheat HD2967 also showed a significant reduction in ATIs content as well as a reduction in α-amylase and trypsin inhibition activity. This result suggests the possibility of developing low immunogenic wheat lines by multiple gene editing for the immunogenic macromolecules.
    Date: 2025-01-30
    Authors: Phogat S, Poudel A, Gayatri, Kaushik M, Madhavan J, Sevanthi AM, Padaria JC, Nekrasov V, Singh PK, Mandal PK.
    Ref: Research Square
  4. ABSTRACT Skin-penetrating nematodes are one of the most prevalent causes of disease worldwide – nearly 15% of the global population is infected with at least one species of skin-penetrating nematode 1,2 . The World Health Organization has targeted these parasites for elimination by 2030 3 , but the lack of preventative measures is a major obstacle to this goal. The infective larvae of skin-penetrating nematodes enter hosts through skin 4 , and blocking skin penetration is an as-yet unexplored approach for preventing infection. However, in order to prevent worm ingress via the skin, an understanding of the behavioral and neural mechanisms that drive skin penetration is required. Here, we describe the skin-penetration behaviors of the human-infective threadworm Strongyloides stercoralis . Using fluorescently labeled worms to enable visualization on the skin coupled with time-lapse microscopy, we show that S. stercoralis engages in repeated cycles of pushing, puncturing, and crawling on the skin surface before penetrating the skin. Pharmacological inhibition of dopamine signaling inhibits these behaviors in S. stercoralis and the human hookworm Ancylostoma ceylanicum, suggesting a critical role for dopamine signaling in driving skin penetration across distantly related nematodes. CRISPR-mediated disruption of dopamine biosynthesis and chemogenetic silencing of dopaminergic neurons also inhibit skin penetration. Finally, inactivation of the TRPN channel TRP-4, which is expressed in the dopaminergic neurons, blocks skin penetration on both rat and human skin. Our results suggest that drugs targeting TRP-4 and other nematode-specific components of the dopaminergic pathway could be developed into topical prophylactics that block skin penetration, thereby preventing infections.
    Date: 2025-01-30
    Authors: Patel R, Romero AG, Bryant AS, Agak GW, Hallem EA.
    Ref: bioRxiv
  5. The metabolic enzyme Aldehyde Dehydrogenase 1A1 (ALDH1A1), a cancer stem cell marker associated with poor outcomes in breast cancer, has emerged as a promising therapeutic target in TNBC. The aim of this study was to investigate the role of ALDH1A1 in radiation resistance and redox stress in TNBC. Functional knockouts of ALDHA1A1 were generated by CRISPR/Cas9-mediated deletion of ALDH1A1 in the SUM159 cell line, and three distinct clonal populations were isolated. Genetic targeting was confirmed by Sanger sequencing, and loss of ALDH1A1 protein expression was validated by Western blotting. Functional assays assessed ALDEFLUOR activity, cell viability, self-renewal capacity, and reactive oxygen species (ROS) levels with or without radiation in both the bulk population and clonal lines. Interestingly, ALDEFLUOR activity was uniformly lost across all clonal lines; however, functional effects of ALDH1A1 loss on redox stress, survival, and radiation sensitivity were observed in only one clonal population. These findings highlight significant variability in the role of ALDH1A1 among clonal populations, reflecting the complexity of tumor heterogeneity. This underscores the importance of accounting for tumor heterogeneity when targeting ALDH1A1, as certain TNBC subpopulations may rely more heavily on ALDH1A1 function. These insights are critical for developing effective ALDH1A1-targeted therapies.
    Date: 2025-01-29
    Authors: Ajayi GO, Ma A, Moadarai SR, Opdenaker LM, Sims Mourtada J.
    Ref: Preprints.org
  6. The interplay between mechanical forces and genetic programs is fundamental to embryonic development, yet how these factors independently or jointly influence morphogenesis and cell fate decisions remains poorly understood. Here, we fine-tune the mechanical environment of murine gastruloids, three-dimensional in vitro models of early embryogenesis, by embedding them in bioinert hydrogels with precisely tunable stiffness and timing of application. This approach reveals that external constraints can selectively influence transcriptional profiles, patterning, or morphology, depending on the level and timing of mechanical modulation. Gastruloids embedded in ultra-soft hydrogels (< 30 Pa) elongate robustly, preserving both anteroposterior patterning and transcriptional profiles. In contrast, embedding at higher stiffness disrupts polarization while leaving gene expression largely unaffected. Conversely, earlier embedding significantly impacts transcriptional profiles independently of polarization defects, highlighting the uncoupling of patterning and transcription. These findings suggest that distinct cellular states respond differently to external constraints. Live imaging and cell tracking imply that impaired cell motility underlies polarization defects, underscoring the role of mechanical forces in shaping morphogenesis independently of transcriptional changes. By allowing precise control over external mechanical boundaries, our approach provides a powerful platform to dissect how physical and biochemical factors interact to orchestrate early embryonic development.
    Date: 2025-01-29
    Authors: Pineau J, Wong-Ng J, Mayran A, Lopez-Delisle L, Osteil P, Shoushtarizadeh A, Duboule D, Gobaa S, Gregor T.
    Ref: bioRxiv
  7. CRISPR-Cas9 is a nuclease creating DNA breaks at sites with sufficient complementarity to the RNA guide. Notably, Cas9 does not require exact RNA-DNA complementarity and can cleave off-target sequences. Various high-accuracy Cas9 variants have been developed, but the precise mechanism of how these variants achieve higher accuracy remains unclear. Here, we develop a kinetic model of Cas9 substrate selection and cleavage. We parameterize the model using datasets available in the literature, including both high-throughput substrate binding and cleavage data and Förster resonance energy transfer measurements of the Cas9 HNH domain transitions. Based on the observed transition statistics, we predict that the Cas9 substrate recognition and cleavage mechanism must allow for HNH domain transitions independent of substrate binding. Additionally, we show that the enhancement in Cas9 substrate specificity must be due to changes in kinetics rather than changes in substrate binding affinities. Furthermore, the fitted model produces quantitatively realistic cleavage error predictions for substrates with protospacer adjacent motif (PAM)-distal mismatches. Finally, we use our model to identify kinetic parameters for HNH domain transitions that can be perturbed to enable high-accuracy cleavage while maintaining cleavage speeds. Our results refine the biophysical mechanism of Cas9 cleavage to inform future routes for its engineering.
    Date: 2025-01-29
    Authors: Hecht AD, Igoshin OA.
    Ref: bioRxiv
  8. In the absence of telomerase, telomere shortening triggers the DNA damage checkpoint and replicative senescence, a potent tumor suppressor mechanism. Paradoxically, this same process is also associated with oncogenic genomic instability. Yet, the precise mechanism that connects these seemingly opposing forces remains poorly understood. To directly study the complex interplay between senescence, telomere dynamics and genomic instability, we developed a system in Saccharomyces cerevisiae to generate and track the dynamics of telomeres of precise length in the absence of telomerase. Using single-telomere and single- cell analyses combined with mathematical modeling, we identify a threshold length at which telomeres switch into dysfunction. A single shortest telomere below the threshold length is necessary and sufficient to trigger the onset of replicative senescence in a majority of cells. At population level, fluctuation assays establish that rare genomic instability arises predominantly in cis to the shortest telomere as non-reciprocal translocations that result in re- elongation of the shortest telomere and likely escape from senescence. The switch of the shortest telomere into dysfunction and subsequent processing in telomerase-negative cells thus serves as the mechanistic link between replicative senescence onset, genomic instability and the initiation of post-senescence survival, explaining the contradictory roles of replicative senescence in oncogenesis.
    Date: 2025-01-29
    Authors: Berardi P, Martinez-Fernandez V, Rat A, Rosas Bringas FR, Jolivet P, Langston R, Mattarocci S, Maes A, Aspert T, Zeinoun B, Casier K, Kazemier HG, Charvin G, Doumic M, Chang M, Teixeira MT.
    Ref: bioRxiv
  9. Glioblastoma (GBM) is a heterogeneous and aggressive brain tumour that is invariably fatal despite maximal treatment. Genetic or transcriptomic ‘biomarkers’ could be used to stratify patients for treatments, however, pairing biomarkers with appropriate therapeutic ‘targets’ is challenging. Consequently, therapeutics have not yet been optimised for specific GBM patient subsets. Here we integrate genome-wide CRISPR/Cas9 knockout screening and genetic-annotation data for 60 distinct patient-derived, IDH wildtype , adult GBM cell lines, quantifying the essentiality of 15,145 genes. We describe a novel method using Targeted Learning, to estimate the effect size of GBM-relevant biomarkers on context-dependent gene essentiality (GBM-CoDE). We derive multiple target-biomarker pair hypotheses, which we release in an accessible platform to accelerate translation to biomarker-stratified clinical trials. Two of these (WWTR1 with EGFR mutation/amplification, and VRK1 with VRK2 expression suppression) have been validated in GBM, implying that our additional novel findings may be valid. Our method is readily translatable to other cancers of unmet need.
    Date: 2025-01-29
    Authors: Foster MT, Ewing A, Frame M, Brennan PM, Khamseh A, Beentjes SV, Carragher NO, Semple CA.
    Ref: bioRxiv
  10. Hypertrophic scar (HS) is a prevalent yet unresolved wound healing complication characterized by persistent hyperactive and proliferative fibroblasts, leading to excessive extracellular matrix (ECM) synthesis and collagen contraction. Our previous studies have identified epidermal stem cells (ESCs) as critical for wound healing and HS remodeling, with its extracellular vesicles (EVs) playing a vital role. However, the specific mechanisms remain unclear. In this study, we first discovered that ESC-EVs could effectively induce the mesenchymal-epidermal transition (MET) of HS fibroblasts (HSFs) and inhibit their biological activity. Furthermore, by next-generation sequencing and multiplexed CRISPR/Cas9 system, we elucidated that this therapeutic effect is mediated by the miR-200 family (miR-200s) encapsulated in ESC-EVs, which targeted and inhibited ZEB1 and ZEB2 in HSFs. This vital role and mechanism have been thoroughly validated in both in vitro cell experiments and in vivo rat tail HS (RHS) models. These findings not only shed light on a previously unidentified mechanism of ESC-EVs for HS, but also provide potential novel targets and strategies for its precise treatment.
    Date: 2025-01-29
    Authors: Zhen M, Xie J, Yang R, Liu L, Liu H, He X, Gao S, Zhu J, Li J, Shu B, Wang P.
    Ref: bioRxiv
  11. Summary Mitochondrial disorders (MDs) are among the most common inborn errors of metabolism and primarily arise from defects in oxidative phosphorylation (OXPHOS). Their complex mode of inheritance and diverse clinical presentations render the diagnosis of MDs challenging and, to date, most lack a cure. Here, we build on previous efforts to discover genes necessary for OXPHOS and report a highly complementary galactose-sensitized CRISPR-Cas9 “growth” screen, presenting an updated inventory now with 481 OXPHOS genes, including 157 linked to MDs. We further focus on FAM136A , a gene associated with Ménière’s disease, and show that it supports inter-membrane space protein homeostasis and OXPHOS in cell lines, mice, and patients. Our study identifies a mitochondrial basis in a familial form of Ménière’s disease (fMD), provides a comprehensive resource of OXPHOS-related genes, and sheds light on the pathways involved in mitochondrial disorders, with the potential to guide future diagnostics and treatments for MDs. Graphical abstract Bullet points Genome-wide CRISPR-Cas9 growth screening complements death screening 481 genes essential for OXPHOS, including 157 mitochondrial disease genes FAM136A supports mitochondrial intermembrane space protein homeostasis Depletion of FAM136A in Ménière’s disease models causes OXPHOS defects
    Date: 2025-01-29
    Authors: Harhai M, Foged MM, Recazens E, Lisci M, Chollet S, Landoni JC, Laban N, Manley S, Lopez-Escamez JA, Lysakowski A, Jourdain AA.
    Ref: bioRxiv
  12. ABSTRACT In public health emergencies or in resource-constrained settings, laboratory-based diagnostic methods, such as RT-qPCR, need to be complemented with accurate, rapid, and accessible approaches to increase testing capacity, as this will translate into better outcomes in disease prevention and management. Here, we develop an original nucleic acid detection platform by leveraging the CRISPR-Cas9 and lateral flow immunochromatography technologies. In combination with an isothermal amplification that runs with a biotinylated primer, the system exploits the interaction between the CRISPR-Cas9 R-loop formed upon targeting a specific nucleic acid and a fluorescein-labelled probe to generate a visual readout on a lateral flow device. Our method enables rapid, sensitive detection of nucleic acids, achieving a limit of 1-10 copies/μL in 1 h at low temperature. We validated the efficacy of the method using clinical samples of patients infected with SARS-CoV-2. Compared to other assays, it operates with more accessible molecular elements and showcases a robust signal-to-noise ratio. Moreover, multiplexed detection was demonstrated using primers labeled with biotin and digoxigenin, achieving simultaneous identification of target genes on lateral flow devices with two test lines. We successfully detected SARS-CoV-2 and Influenza A (H1N1) in spiked samples, highlighting the potential of the method for multiplexed diagnostics of respiratory viruses. All in all, this represents a versatile and manageable platform for point-of-care testing, thereby supporting better patient outcomes and enhanced pandemic preparedness.
    Date: 2025-01-28
    Authors: Montagud-Martínez R, Márquez-Costa R, Ruiz R, Martínez-Aviñó A, Ballesteros-Garrido R, Navarro D, Campins-Falcó P, Rodrigo G.
    Ref: medRxiv
  13. Abstract Backgrounds and Aim: Colorectal cancer (CRC) pathogenesis is correlated with dysregulation of tight junction. This study aimed to investigate the molecular mechanism by which trimethylamine N-oxide (TMAO) alters the expression of tight junction proteins in a colorectal cancer (CRC) cell line. Material and Method: The study utilized the CRISPR/Cas13 system for targeted knock down of HULC in Caco-2 cells, followed by treatment with trimethylamine N-Oxide (TMAO). Tight junction components, including ZO-1, Claudin-1, and Occludin, were analyzed using real-time quantitative polymerase chain reaction (RT-qPCR). To investigate the role of the P38MAPK pathway, the specific inhibitor SB203580 was used in cells treated with TMAO to comprehensively assess tight junction regulation. Statistical analysis was performed using one-way ANOVA to compare the mean ± SD between different groups, followed by paired comparisons using the t-test. Results: Cells treated with TMAO showed a significant upregulation of the oncogenic long non-coding RNA (lncRNA) HULC (Highly Upregulated in Liver Cancer), , accompanied by increased expression of p38 MAPK. Interestingly, a significant downregulation of ZO-1 and Claudin-1 was observed as a result of TMAO treatment, which was modulated by the HULC/p38 MAPK axis. However, Occludin expression was also reduced by TMAO, but it remained unaffected by the HULC/p38 MAPK pathway. Conclusion: This study revealed a novel TMAO/HULC/p38 MAPK axis involved in the regulation of tight junctions in a colorectal cancer cell line model. TMAO treatment significantly reduced the expression of ZO-1, Claudin-1, and Occludin. Further in vivo research is strongly recommended to clarify the impact of TMAO on the integrity of colorectal cancer cells.
    Date: 2025-01-28
    Authors: Najafpour S, Moradzad M, Rahimi K, Alighardashi Z, Vahabzadeh Z.
    Ref: Research Square
  14. The second messenger bis -(3′→5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) governs adaptive responses in the opportunistic pathogen Pseudomonas aeruginosa , including biofilm formation and the transition from acute to chronic infections. Understanding the intricate c-di-GMP signaling network remains challenging due to the overlapping activities of numerous diguanylate cyclases (DGCs). In this study, we employed a CRISPR-based multiplex genome-editing tool to disrupt all 32 GGDEF domain-containing proteins (GCPs) implicated in c-di-GMP signaling in P . aeruginosa UCBPP-PA14. Phenotypic and physiological analyses revealed that the resulting mutant was unable to form biofilms and had attenuated virulence. Residual c-di-GMP levels were still detected despite the extensive GCP disruption, underscoring the robustness of this regulatory network. Taken together, these findings provide insights into the complex c-di-GMP metabolism and showcase the importance of functional overlapping in bacterial signaling. Moreover, our design overcomes the native redundancy in c-di-GMP synthesis, providing a framework to dissect individual DGC functions and paving the way for targeted strategies to address bacterial adaptation and pathogenesis.
    Date: 2025-01-28
    Authors: Martino RA, Volke DC, Tenaglia AH, Tribelli PM, Nikel PI, Smania AM.
    Ref: bioRxiv
  15. Cultivated oat ( Avena sativa ) is an emerging cereal for healthy lives owing to its unique characteristics, such as high β-glucan and oil content, distinctive fatty acid composition, and gluten-free nature. The recent unravelling of the 12.5 Gb hexaploid oat genome underlined breeding barriers caused by ancestral translocations and inversions, leading to recombination suppression and pseudo-linkage further hindering conventional trait introgression. Over the past decade, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system has been extensively used for crop improvement and functional genomics in all other cereals except oats. Its large repetitive genome with three sub-genomes, lack of efficient transformation, recalcitrant nature, and complex molecular screening due to gene redundancy have been major obstacles to gene editing success in oat. We report the first successful CRISPR-Cas9-based gene editing in oat in three genes — AsTLP8, AsVRN3 and AsVRN3D with gene-editing efficiency of up to 41.1%. The gene-edited plants for all the genes carried deletions and/or one base insertion. Further analysis of VRN3 T 1 and T 2 mutants revealed bent leaves in heterozygous knockouts (AACCdD), while an extended vegetative growth phase was seen in the T 1 homozygous and biallelic mutants (aaccdd), accentuating the important role of VRN3 in oat development. We are confident that this highly efficient oat gene editing system will pave the way for a deeper molecular understanding of this healthy cereal, deciphering oat’s functional genomics, and creating genetic diversity at the cold spots of recombination in oat.
    Date: 2025-01-28
    Authors: Mehtab-Singh, Kaye C, Kaur R, Singh J.
    Ref: bioRxiv
  16. ABSTRACT Arthropods have an incredible diversity of limbs that are modified for walking, chewing, cleaning, mating, grasping, sensing, and more. Understanding the relationships and evolutionary histories of different limbs is a central task, but their sheer diversity makes this a daunting if not impossible task using morphology alone. Here, the in situ expression patterns and CRISPR-Cas9 phenotypes for the five best-studied leg-patterning genes – Distal-less , Sp6-9 , dachshund , extradenticle , and homothorax – are described for all limbs of the crustacean Parhyale . Crustaceans are well-suited for this task because their limbs are more diverse than those of other arthropods, and each individual possesses a wide range of limb types that are relevant to many other arthropods, living and extinct. These results will a) provide a template for understanding the genetic basis of limb construction in arthropods more generally based on the strong phenotypes that can be obtained with CRISPR-Cas9, and b) contribute to our understanding of the evolution and affinities of highly modified legs like mouthparts and genitalia using molecular methods to complement previous morphological and embryological approaches.
    Date: 2025-01-28
    Authors: Bruce HS, Patel NH.
    Ref: bioRxiv
  17. Motivation Understanding the factors involved in DNA double-strand break (DSB) repair is crucial for the development of targeted anti-cancer therapies, yet the roles of many genes remain unclear. Recent studies show that perturbations of certain genes can alter the distribution of sequence-specific mutations left behind after DSB repair. This suggests that genome-wide screening could reveal novel DSB repair factors by identifying genes whose perturbation causes the mutational distribution spectra observed at a given DSB site to deviate significantly from the wild-type. However, designing proper controls for a genome-wide perturbation screen could be challenging. We explore the idea that a genome-wide screen might allow us to forgo the use of traditional non-targeting controls by reframing the analysis as an outlier detection problem, assuming that most genes have minimal influence on DSB repair. Results We propose MUSICiAn (Mutational Signature Catalogue Analysis), a compositional data analysis method that ranks gene perturbation-specific mutational spectra without controls by measuring deviations from the central tendency in the distributions of all spectra. We show that MUSICiAn can effectively estimate pseudo-controls for the existing Repair-seq dataset, screening 476 genes and 60 non-targeting controls. We further apply MUSICiAn to a genome-wide dataset profiling mutational outcomes induced by CRISPR-Cas9 at three target sites across cells with individual perturbations of 18,406 genes. MUSICiAn successfully recovers known genes, highlights the spliceosome as a lesser-appreciated player in DSB repair, and reveals candidates for further investigation. Availability github.com/joanagoncalveslab/MUSICiAn .
    Date: 2025-01-28
    Authors: Seale C, Barazas M, van Schendel R, Tijsterman M, Gonçalves JP.
    Ref: bioRxiv
  18. The maize smut fungi Ustilago maydis and Sporisorium reilianum are closely related and have similar genomes in terms of size and synteny. While U. maydis induces tumors locally at sites of infection, S. reilianum systemically colonizes the host and causes symptoms in the inflorescences. To investigate the genetic basis of these differences, an interspecific recombinant hybrid (rUSH) with the mating type system of S. reilianum was generated. rUSH exhibited extensive in-planta proliferation, showing a S. reilianum -like phenotype at all developmental stages except teliospore formation. Transcriptome profiling revealed that expression of pathogenicity-related effector gene orthologs was induced in rUSH, but not in a wild-type hybrid control. Multiple transcriptome comparisons identified 253 differentially expressed one-to-one effector orthologs with distinct regulatory patterns, including cis-, trans-, and rUSH-specific regulation. Functional analysis via CRISPR/Cas9 mutagenesis uncovered three novel virulence factors among the rUSH-specific regulated effectors. Ultimately, rUSH facilitated to identify the transcription factor UmHdp2 as key regulator of U. maydis- induced tumorigenesis. Together, these findings highlight the utility of a recombinant, interspecific hybrid in unraveling the molecular mechanisms underlying pathogenic differences in closely related fungal pathogens.
    Date: 2025-01-28
    Authors: Werner J, Zuo W, Winkler T, Doehlemann G.
    Ref: bioRxiv
  19. CRISPR base editors are crucial for precise genome manipulation. Existing APOBEC-based cytosine base editors (CBEs), while powerful, exhibit indels and sequence context limitations, where TC-context preferences restrict effective editing of CC and GC motifs. To address these challenges, we evaluated various tRNA adenine deaminase (TadA)-derived CBEs, ultimately engineering zTadCBE that demonstrates high editing efficiency, minimized off-target effects, and an expanded targeting range. Our approach integrates beneficial mutations from TadA-based adenine base editors (ABEs) with SpRYCas9n-enhanced protospacer-adjacent motif (PAM) compatibility. Additionally, we engineered expanded-window zTadCBE variants, zTadCBE-ex1 and zTadCBE-ex2, to target wider nucleotide ranges, further increasing the versatility of this tool. To demonstrate the utility of zTadCBE variants in the functional assessment of genetic mutants, we generated a model for CDH23-associated hearing loss to validate the pathogenicity of a patient-specific variant in zebrafish. Furthermore, we induced a premature stop codon in the mediator complex gene med12 to inactivate its function using a CRISPR-STOP strategy and recapitulated patient-specific phenotypes in the founding (F0) generation. zTadCBE variants thus offer a robust set of CBEs for precise and efficient C-to-T editing in zebrafish, promising to advance the rapid functional assessment of genetic variants in vivo.
    Date: 2025-01-28
    Authors: Qin W, Lin S, Petree C, Varshney P, Varshney GK.
    Ref: bioRxiv
  20. Examining early-branching animal phyla can help reconstructing the evolutionary origins of immune cells. Here, we characterized immune-related cell programs in embryos of the sea anemone Nematostella vectensis, a model of Cnidaria, which diverged ~600 million years ago from other animals. Using a transgenic Nematostella reporter line expressing mCherry under the RLRb antiviral promoter, we identified a morphologically and transcriptomically distinct cell population activated by the viral mimic poly(I:C). These cells upregulate immune effector and regulator genes and show increased phagocytic activity. Bulk RNA sequencing of RLRb expressing cells and single-cell transcriptomics revealed gene regulatory programs expressed in specialized immune cells under basal conditions and upon activation. Comparing the Nematostella immune expression profile with that of stony corals treated with the immunostimulant 2'-3'-Cyclic GMP-AMP demonstrated a conserved immune response across Hexacorallia. This study uncovers a novel cnidarian immune cell type involved in antiviral immunity, providing insights into the evolutionary history of innate immunity.
    Date: 2025-01-27
    Authors: Kozlovski I, Sharoni T, Levy S, Jaimes-Becerra A, Talice S, Kwak H, Aleshkina D, Aharoni R, Grau-Bove X, Karmi O, Rosental B, Sebe-Pedros A, Moran Y.
    Ref: bioRxiv
  21. INTRODUCTION Africa, home to 1.4 billion people and the highest genetic diversity globally, harbors unique genetic variants crucial for understanding complex diseases like neurodegenerative disorders. However, African populations remain underrepresented in induced pluripotent stem cell (iPSC) collections, limiting the exploration of population-specific disease mechanisms and therapeutic discoveries. METHODS To address this gap, we established an open-access African Somatic and Stem Cell Bank. RESULTS In this initial phase, we generated 10 rigorously characterized iPSC lines from fibroblasts representing five Nigerian ethnic groups and both sexes. These lines underwent extensive profiling for pluripotency, genetic stability, differentiation potential, and Alzheimer’s disease and Parkinson’s disease risk variants. CRISPR/Cas9 technology was used to introduce frontotemporal dementia-associated MAPT mutations (P301L and R406W). DISCUSSION This collection offers a renewable, genetically diverse resource to investigate disease pathogenicity in African populations, facilitating breakthroughs in neurodegenerative research, drug discovery, and regenerative medicine.
    Date: 2025-01-27
    Authors: Maina MB, Isah MB, Marsh JA, Muhammad Z, Babazau L, Idris AA, Aladyeva E, Miller N, Starr E, Miller KJ, Lee S, Minaya M, Wray S, Harari O, Goni BW, Serpell LC, Karch CM.
    Ref: medRxiv
  22. Microproteins, short functional peptides encoded by small genes, are emerging as critical regulators of cellular processes, yet their roles in mitochondrial function and neurodegeneration remain underexplored. In this study, we identify NCBP2-AS2 as an evolutionarily conserved mitochondrial microprotein with significant roles in energy metabolism and neurogenesis. Using a combination of cellular and molecular approaches, including CRISPR/Cas9 knockout models, stoichiometric co- immunoprecipitation, and advanced imaging techniques, we demonstrate that NCBP2-AS2 localizes to the inner mitochondrial space and interacts with translocase of the inner membrane (TIM) chaperones. These interactions suggest a role in ATPase subunit transport, supported by the observed reductions in ATPase subunit levels and impaired glucose metabolism in NCBP2-AS2-deficient cells. In zebrafish, NCBP2-AS2 knockout led to increased astroglial proliferation, microglial abundance, and enhanced neurogenesis, particularly under amyloid pathology. Notably, we show that NCBP2-AS2 expression is consistently downregulated in human Alzheimer’s disease brains and zebrafish amyloidosis models, suggesting a conserved role in neurodegenerative pathology. These findings reveal a novel link between mitochondrial protein transport, energy metabolism, and neural regeneration, positioning NCBP2-AS2 as a potential therapeutic target for mitigating mitochondrial dysfunction and promoting neurogenesis in neurodegenerative diseases such as Alzheimer’s disease.
    Date: 2025-01-27
    Authors: Popova S, Bhattarai P, Yilmaz E, Lascu D, Kuo J, Erdem G, Coban B, Michling J, Cosacak MI, Tayran H, Kurth T, Schambony A, Buchholz F, Gentzel M, Kizil C.
    Ref: bioRxiv
  23. ABSTRACT Fibrosis and persistent inflammation are interconnected processes that inhibit axon regeneration in the mammalian central nervous system (CNS). In zebrafish, by contrast, fibroblast-derived extracellular matrix deposition and inflammation facilitate regeneration. However, the regulatory cross-talk between fibroblasts and the innate immune system in the regenerating CNS is not understood. Here, we show that zebrafish fibroblasts possess a dual role in inducing and subsequently resolving inflammation, which are both essential for regeneration. We identify a transient, injury-specific cthrc1a + fibroblast state with an inflammation-associated, less differentiated, and non-fibrotic profile. Induction of this fibroblast state precedes and contributes to the initiation of the inflammatory response. At the peak of neutrophil influx, cthrc1a + fibroblasts coordinate the resolution of inflammation. Disruption of these inflammation dynamics inhibits axon regeneration and alters the mechano-structural properties of the lesion environment. This establishes the biphasic inflammation control by dedifferentiated fibroblasts as a pivotal mechanism for CNS regeneration. ONE SENTENCE SUMMARY Dedifferentiated fibroblasts sequentially induce and resolve neutrophil-driven inflammation through cytokine release to facilitate axon regeneration after spinal cord injury in zebrafish. HIGHLIGHTS Time-resolved single-cell transcriptomics of zebrafish spinal cord regeneration. Spinal cord injury induces fibroblast dedifferentiation. Dedifferentiated fibroblasts sequentially induce and resolve inflammation. Dysregulation of inflammation dynamics alters mechano-structural tissue properties.
    Date: 2025-01-27
    Authors: John N, Fleming T, Kolb J, Lyraki O, Vásquez-Sepúlveda S, Parmar A, Kim K, Tarczewska M, Gupta P, Singh K, Marini F, Singh SP, Falk S, Franze K, Guck J, Wehner D.
    Ref: bioRxiv
  24. Small noncoding RNAs (smRNAs) play critical roles in regulating various cellular processes, including development, stress response, and disease pathogenesis. However, functional characterization of smRNAs remains limited by the scale and simplicity of phenotypic readouts. Recently, single-cell perturbation screening methods, which link CRISPR-mediated genetic perturbations to rich transcriptomic profiling, have emerged as foundational and scalable approaches for understanding gene functions, mapping regulatory networks, and revealing genetic interactions. However, a comparable approach for probing the regulatory consequences of smRNA perturbations is lacking. Here, we present Decoy-seq as an extension of this approach for high-content, single-cell perturbation screening of smRNAs. This method leverages U6-driven tough decoys (TuD), which form stable duplexes with their target smRNAs, for inhibition in the cell. Lentiviral-encoded TuDs are compatible with conventional single-cell RNA-sequencing (scRNA-seq) technologies, allowing joint identification of the smRNA perturbation in each cell and its associated transcriptomic profile. We applied Decoy-seq to 336 microRNAs (miRNAs) and 196 tRNA-derived fragments (tRFs) in a human breast cancer cell line, demonstrating its ability to uncover complex regulatory pathways and novel functions of these smRNAs. Notably, we show that tRFs influence mRNA polyadenylation and regulate key cancer-associated processes, such as cell cycle progression and proliferation. Therefore, Decoy-seq provides a powerful framework for exploring the functional roles of smRNAs in normal physiology and disease, and holds promise for accelerating future discoveries.
    Date: 2025-01-27
    Authors: Choi B, Sobti S, Soto LM, Charbonneau T, Sababi A, Navickas A, Najafabadi HS, Goodarzi H.
    Ref: bioRxiv
  25. Latent EBV infection is causally associated with various B-cell malignancies, while periodic lytic-cycle replication is essential for sustaining viral progeny. Lytic cycle induction represents a promising therapeutic strategy for EBV-associated neoplasms. Therefore, uncovering the mechanisms that regulate EBV lytic-cycle reactivation is pivotal for understanding viral pathogenesis and advancing novel therapies. Our genome-wide transcriptomic analysis reveals that E2F1 expression is transcriptionally activated during EBV latent infection in B-lymphocytes but significantly suppressed during lytic cycle reactivation. While ectopic E2F1 expression suppresses lytic replication, E2F1 depletion markedly accelerates this process. Mechanistically, we establish that E2F1 and the lytic transactivator BZLF1 form a negative transcriptional feedback loop, tightly controlling viral lytic replication. Furthermore, E2F1 positively regulates c-Myc expression and together they repress the leaky BZLF1 expression during latency. Importantly, c-Myc does not influence E2F1 expression, nor does BZLF1 modulate c-Myc transcription, underlining a distinct regulatory hierarchy. In sum, our findings reveal that EBV tightly controls the latent-to-lytic switch through precise regulation of E2F1 expression, positioning E2F1 as a pivotal regulator of both cellular and viral gene expression. Synopsis EBV coordinates the latent-to-lytic switch by sensing E2F1 abundance, which acts as a crucial transcriptional regulator of both cellular and viral gene expressions. During EBV latent infection, E2F1 promotes c-Myc transcription, and together they suppress EBV lytic cycle transactivator BZLF1 expression. E2F1 and BZLF1 form a negative feedback loop in order to control each other’s transcriptions. BZLF1-driven controlled E2F1 expression successively inhibits c-Myc level, thereby stimulating EBV lytic cycle reactivation. BZLF1 does not regulate c-Myc, nor does c-Myc reciprocally regulate E2F1, emphasizing a unidirectional regulatory hierarchy.
    Date: 2025-01-26
    Authors: Biswas J, Ali SA, Malik S, Nag S, Mukherjee P, Saha A.
    Ref: bioRxiv
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