Results 1-25 of about 1,000
  1. TYR encodes tyrosinase, the enzyme catalysing the initial steps of melanin biosynthesis in melanocytes and retinal pigment epithelia (RPE). TYR c.1205G>A (p.Arg402Gln) is a common genetic variant associated with several pigmentation traits. Notably, when this variant is encountered in specific haplotypic backgrounds in the homozygous state, it predisposes to albinism. We generated an induced pluripotent stem cell (iPSC) line from an affected individual carrying such a homozygous genotype (UMANi255-A), and then used CRISPR-Cas9 to correct the TYR c.1205G>A variant (UMANi255-A-1). The resulting iPSC lines demonstrate capacity for multi-lineage differentiation, providing a useful in vitro model for studying pigmentation biology.
    Date: 2025-07-16
    Authors: Downton P, Bates N, Woods S, Adamson A, Sergouniotis PI.
    Ref: bioRxiv
  2. Abstract Background - The root-knot nematode Meloidogyne incognita , is a highly destructive parasite that manipulates host plant processes through effector proteins, affecting agriculture globally. Despite advances in genomic and transcriptomic studies, the regulatory mechanisms controlling effector gene expression, especially at the chromatin level, are still poorly understood. Gene regulation studies in plant-parasitic nematodes (PPN) face several challenges, including the absence of transformation systems and technical barriers in chromatin preparation, particularly for transcription factors (TFs) expressed in secretory gland cells. Conventional methods like Chromatin Immunoprecipitation (ChIP) are limited in PPN due to low chromatin yields, the impermeability of nematode cuticles, and difficulties in producing antibodies for low-abundance TFs. These issues call for alternative approaches, such as dCas9-based CAPTURE (CRISPR Affinity Purification in siTU of Regulatory Elements) that allows studying chromatin interactions by using a catalytically inactive dCas9 protein to target specific genomic loci without relying on antibodies. Results - This study presents an optimized in vitro dCas9-based CAPTURE for M. incognita that addresses key challenges in chromatin extraction and stability. The protocol focuses on the promoter region of the effector gene 6F06 , a critical gene for parasitism. Several optimizations were made, including improvements in nematode disruption, chromatin extraction, and protein-DNA complex stability. This method successfully isolated chromatin-protein complexes and identified four putative chromatin-associated proteins, including BANF1, linked to chromatin remodelling complexes like SWI/SNF. Conclusion - The optimized in vitro dCas9-based CAPTURE protocol offers a new tool for investigating chromatin dynamics and regulatory proteins in non-transformable nematodes. This method expands the scope of effector gene regulation research and provides new insights into parasitism in M. incognita . Future research will aim to validate these regulatory proteins and extend the method to other effector loci, potentially guiding the development of novel nematode control strategies.
    Date: 2025-07-16
    Authors: Bournaud C, Tollec A, Danchin EGJ, Couté Y, Akker SE.
    Ref: Research Square
  3. Pathological accumulation of four-repeat (4R) tau is central to several frontotemporal dementia (FTD) subtypes but human neuronal models amenable to high-throughput screening of 4R tau-targeting therapies remain limited. To address this gap, we developed iPSC-derived i3Neuron (i3N) lines expressing >75% 4R tau, driven by FTD splice-shifting mutations (S305N or S305N/IVS10+3). These neurons develop hyperphosphorylated tau and demonstrate somatodendritic mis-localisation. Unlike any other stem cell model of 4R tauopathy, these i3N neurons develop endogenous seed-competent tau and present pFTAA-positive tau assemblies after 28 days in culture. For scalable screening, we CRISPR-engineered a HiBiT luminescence tag at the endogenous MAPT locus into the S305N/IVS10+3 iPSC line, enabling precise quantification of tau levels and pharmacological responses. The model responded predictably to compounds affecting tau clearance, demonstrating its suitability for drug discovery. Overall, this i3N platform recapitulates key features of 4R tauopathy and provides a robust system to identify therapeutic modulators of pathological tau.
    Date: 2025-07-15
    Authors: Tsefou E, Bez S, Birkle TJ, Foiani M, Watamura N, Bourdenx M, Gavriouchkina D, Turkes E, Crawford S, Coneys R, Isaacs A, Duff K.
    Ref: bioRxiv
  4. Flaviviruses are genetically related, yet cause distinct disease patterns ranging from hepatitis and vascular shock syndrome to encephalitis and congenital abnormalities. There is an incomplete understanding of the cellular pathways co-opted by flaviviruses, and differences in host response to infection may underlie the diverse pathologies caused. We present a single-cell approach (Quantification of Infection and CRISPR guide sequencing; QIC-seq) that combines CRISPR/Cas9 knockout with virus-inclusive transcriptomics to systematically compare host factor requirements and host transcriptional response to flaviviral challenge. We show that dengue and yellow fever viruses are strictly dependent on subunits of the oligosaccharyltransferase complex, while the more distantly related West Nile and Langat viruses are dependent on components of the ER-associated degradation machinery. Our data further shows virus-induced upregulation of interferon-stimulated genes, and activation of the unfolded protein response. Together, QIC-seq enables quantitative comparisons of viral host factor utilization, which may inform development of host-directed antiviral therapies.
    Date: 2025-07-15
    Authors: Dupzyk A, Waldman B, Zengel J, Carette JE.
    Ref: bioRxiv
  5. Abstract Tumor cells acquire survival advantages and evade therapeutic-induced cell death through continuous evolution. The liver has a unique immune microenvironment, with a higher proportion of natural killer (NK) cells compared to other organs. While NK cells are closely associated with liver precancerous conditions and liver cancer, their role in liver cancer evolution remains unclear. This study aims to comprehensively reveal core regulatory factors and key molecular events in liver cancer evolution, and develop novel therapeutic strategies. In this study, Immune-humanized spatiotemporal models were established to simulate liver cancer evolution. Multi-omics approaches, including scRNA-seq, snRNA-seq, spatial transcriptome and CRISPR/Cas9 screening, were subsequently used to establish the functional relevance of NK cells and liver cancer evolution. We demonstrated that early immunosurveillance induced by NK cells is the pioneering power to trigger transition in tumor cell state and impedes subsequent adaptive immunosurveillance. Mechanistically, NK cells induced the reprogramming of lipid metabolism, especially cholesterol accumulation in tumor cells, and tumor stemness enhancement, which are demonstrated to be crucial for liver cancer evolution. Furthermore, combining anti-LAG-3 and LXR activation halts tumor evolution, enhancing the efficacy and durability of immune checkpoint inhibitors against advanced liver cancer. In conclusion, NK cell-mediated early immunosurveillance drives liver cancer evolution. Immunometabolic therapy targeting these evolved tumor cells presents a promising strategy for advanced liver cancer.
    Date: 2025-07-15
    Authors: Shi L, Liu B, Sun R, He J, Shen H, Li W, Wang Y, Zhao W, Jin C, Jin B, Tian Y, Ma L, Hou L, Chen M, Shen J, Shen B, Liang X, Yu H, Wang Y, Wang D, Cai X.
    Ref: Research Square
  6. Background: Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disease caused by mutations in the DMD gene, leading to the absence or dysfunction of dystrophin. While cardiac and skeletal muscles are both affected, tissue-specific differences in disease manifestation and dystrophin regulation remain poorly understood. Methods: To investigate these differences, we established a human induced pluripotent stem cell (hiPSC) model of DMD from peripheral blood mononuclear cells (PBMC) of a patient carrying a splice-site mutation in intron 68 (c.9975-1G>T). An isogenic control line was generated via CRISPR/Cas9 correction. Both repaired and DMD hiPSCs were differentiated into cardiomyocytes (hiPSC-CMs) and skeletal muscle cells (hiPSC-SMs). Transcript and protein analyses were performed, along with functional assessment using microelectrode array. Results: Transcript analysis revealed an in-frame deletion of two amino acids (Tyr3325 and Arg3326) due to skipping of the first six nucleotides of exon 69. Despite this, near full-length Dp427 was detected by western blot, along with expression of Dp116 in hiPSC-CMs. Dystrophin levels were preserved in DMD hiPSC-CMs but markedly reduced in hiPSC-SMs, suggesting tissue-specific regulation. Functional analysis showed altered ?-adrenergic responsiveness in DMD hiPSC-CMs, with increased beating frequency and accelerated repolarization upon isoproterenol stimulation. Conclusions: Our study identifies a splice-site mutation that preserves high level of dystrophin expression in cardiac but reduced in skeletal muscle and reveals Dp116 expression in cardiomyocytes. These findings highlight the importance of tissue context in DMD and demonstrate the power of hiPSC-based systems for dissecting mutation-specific effects.
    Date: 2025-07-15
    Authors: Dulak J, Białobrzeska M, Przymuszała M, Potulska-Chromik A, Kostera-Pruszczyk A, Stępniewski J, Florczyk-Soluch U.
    Ref: medRxiv
  7. Addiction to nicotine and alcohol continues to be a leading cause of death and loss of productivity as measured in disability-adjusted life years. Polymorphisms in the nicotinic acetylcholine receptor subunit α5 (CHRNA5) have been identified as risk factors associated with nicotine dependence in human genetic studies and rodent models. Whether the chrna5 function is also important for phenotypes associated with comorbid disorders independently is a question of interest. We generated a stable mutant line in zebrafish using the CRISPR/Cas-9 technique. We found that the chrna5 mutant fish exhibit an increased acute preference to both nicotine and alcohol in the Self-Administration Zebrafish Assay (SAZA). When subjected to multi-day exposures to either, chrna5 mutants exhibited greater behavioural change, but reduced transcriptomic changes compared to wild type siblings, suggesting an impaired homeostatic regulation following drug exposure. Further, chrna5 mutants exhibited drug-independent changes in appetite and circadian rhythms, suggesting a genetic predisposition to disorders often comorbid with substance dependence. We expect these results to give new insights into the operation of genes whose normal function modulates vulnerability to multi-substance use and comorbid disorders.
    Date: 2025-07-15
    Authors: Goel T, Raine J, Kibat C, Collado JW, Banerjee TD, Mathuru A.
    Ref: bioRxiv
  8. Introduction Angelman Syndrome (AS) is characterized in large part by the loss of functional UBE3A protein in mature neurons. A majority of AS etiologies is linked to deletion of the maternal copy of the UBE3A gene and epigenetic silencing of the paternal copy. A common therapeutic strategy is to unsilence the intact paternal copy thereby restoring UBE3A levels. Identifying novel therapies has been aided by a UBE3A-YFP reporter mouse model. This study presents an analogous fluorescent UBE3A reporter system in human cells. Methods Previously derived induced Pluripotent Stem Cells (iPSCs) with a Class II large deletion at the UBE3A locus are used in this study. mGL and eGFP are integrated downstream of the endogenous UBE3A using CRISPR/Cas9. These reporter iPSCs are differentiated into 2D and 3D neural cultures to monitor long-term neuronal maturation. Green fluorescence dynamics are analyzed by immunostaining and flow cytometry. Results The reporter is successfully integrated into the genome and reports paternal UBE3A expression. Fluorescence expression gradually reduces with UBE3A silencing in neurons as they mature. Expression patterns also reflect expected responses to molecules known to reactivate paternal UBE3A . Discussion This human-cell-based model can be used to screen novel therapeutic candidates, facilitate tracking of UBE3A expression in time and space, and study human-specific responses. However, its ability to restore UBE3A function cannot be studied using this model. Further research in human cells is needed to engineer systems with functional UBE3A to fully capture the therapeutic capabilities of novel candidates.
    Date: 2025-07-14
    Authors: Kelkar GR, Stuppy SR, Sen D, Yagci ZB, Han L, Land L, Hartman JK, Keung AJ.
    Ref: bioRxiv
  9. Polyamines (PAs) are essential for plant development and stress responses, requiring tight homeostatic regulation. Many PA enzymes are regulated post-transcriptionally, making traditional transcript-based methods ineffective in determining their abundance, highlighting the need for alternative approaches to study PA homeostasis. Here, we refined a liquid chromatography mass spectrometry (LC MS) based method to simultaneously quantify activities of two key PA synthesizing enzymes - arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) - from plant tissues using stable isotope substrates. By optimizing substrate concentrations, we increased assay sensitivity >10-fold in tomato leaf tissue. We further adapted this protocol for Nicotiana benthamiana, a model plant widely used for transient recombinant protein expression. Expression of epitope-tagged ADCs in this system revealed a direct correlation between protein abundance and enzymatic activity, demonstrating that ADC activity can infer its protein abundance in native tissues. Proof-of-principle experiments with the N. benthamiana expression system, confirm substrate specificity of tomato ADC and ODC enzymes and essential catalytic residues of tomato ADCs. Beyond enzymatic activities, our LCMS-based method also permits quantification of 11 PA network metabolite concentrations from the same LCMS sample. Visualizing this data as a heatmap pathway diagram, alongside ADC/ODC activities provides a comprehensive overview of PA metabolism in plant tissues. We also studied tomato CRISPR-Cas9-induced mutants deficient in ADC or ODC, complemented by phenotypic analysis. LC-MS analysis of an adc1/adc2 double mutant - an embryo lethal genotype in Arabidopsis - had no detectable agmatine, the product of ADCs. Additionally, despite a reduction in putrescine, no impact on the downstream PAs, spermidine and spermine, was found. The adc1/adc2 double mutant showed severe developmental abnormalities, including complete flower loss, demonstrating the indispensable role of ADCs in flower development. In summary, our optimized LC-MS approach for simultaneous quantification of ADC/ODC enzyme activity and PA-pathway metabolites, the ability to transiently express and functionally analyze recombinant ADC/ODC proteins in planta, and a collection of tomato CRISPR mutants deficient in these enzymes collectively establish a versatile new experimental toolkit to dissect PA homeostasis and PA-dependent developmental processes in plants.
    Date: 2025-07-14
    Authors: Ritchie ES, von Roepenack Lahaye E, Perrett D, Wu D, Lahaye T.
    Ref: bioRxiv
  10. In chordate embryos, placodes are ectodermal thickenings around the borders of the neural plate that give rise to various sensory organs and cell types. While generally thought to be a vertebrate-specific innovation, homologous placodes are proposed to exist in non-vertebrate chordates as well. In Ciona robusta, a solitary tunicate, the adult mouth (the oral siphon) is derived from one such "cranial-like" placode in the larva, which we term the oral siphon placode (OSP). At embryonic and larval stages, the OSP consists of a small rosette of cells that forms from the neuropore at the anteriormost extent of neural tube closure. While the morphogenesis of the OSP and its physical separation from other surface ectoderm structures have been described in detail, how this is regulated at the molecular level is currently unknown. Here we show the involvement of protocadherin-mediated cell-cell adhesion in the segregation and structural cohesiveness of the OSP. Protocadherin.e (Pcdhe.e) is expressed specifically in the OSP but not in other surface ectoderm cells. CRISPR/Cas9-mediated disruption of Pcdh.e in these cells results in loss of OSP structural integrity and ability to physically separate from other structures derived from the same cell lineage. Overexpression of Pcdh.e throughout the anterior surface ectoderm results in similar loss of a physically separate and distinct OSP territory. Furthermore, we show that Pcdh.e expession in the OSP depends on oral placode-specific transcription factors such as Six1/2 and Pitx. Our results suggest that OSP integrity and morphogenesis require precise regulation of a homotypic cell-cell adhesion molecule, which might reflect a conserved mechanism for placode formation in chordates.
    Date: 2025-07-14
    Authors: Vedurupaka S, Jadali B, Johnson CJ, Stolfi A, Popsuj S.
    Ref: bioRxiv
  11. Microalgae induce a CO 2 -concentrating mechanism (CCM) to maintain photosynthesis when dissolved CO 2 is limited, but how this energy-intensive system is suppressed when CO 2 levels rise has remained unclear. The CCM consumes 15–30% of photosynthetically-generated ATP, making its regulation critical for cellular energy balance. Here, we identify a nuclear repressor of the CCM in the green alga Chlamydomonas reinhardtii . A pull-down screen for interacting partners of the master activator CCM1/CIA5 revealed an uncharacterized protein that co-purifies with CCM1 even after high-salt washes. This protein, designated CCM1-binding protein 1 (CBP1), combines a CobW/CobW_C GTP-binding metallochaperone module with a WW domain characteristic of protein-protein interactions. CBP1 colocalizes with CCM1 in the nucleus regardless of CO 2 conditions, and the two proteins interact in vivo . CRISPR/Cas9-based disruption of CBP1 does not affect growth or CCM induction under CO 2 limitation but derepresses 27 of 41 CCM1-dependent low-CO 2 inducible genes under high-CO 2 conditions. These include the periplasmic and intracellular carbonic anhydrases (CAH1, LCIB) and inorganic carbon transporters/channels (LCIA, LCI1, BST1, BST3). Consistently, cbp1 mutants accumulate higher levels of CAH1 and LCIB proteins and exhibit a 40% increase in inorganic carbon affinity under high-CO 2 conditions; this phenotype is rescued by CBP1 complementation or by acetazolamide treatment. These results demonstrate that CBP1 prevents unnecessary CCM activity when CO 2 is abundant, acting upstream of both transporter/channel and carbonic anhydrase modules. Our findings suggest a regulatory mechanism potentially linking zinc-dependent protein chemistry to CCM gene repression, providing insights into energy-efficient CO 2 sensing in aquatic photosynthetic organisms. Significance statement Algae flourish by activating a CO 2 -concentrating mechanism (CCM) when dissolved CO₂ is limited but must deactivate it when CO 2 levels increase to conserve energy. We have identified the nuclear protein that functions as this long sought “off switch” in the model green alga. Deletion of this protein causes cells to overproduce CCM transporters and enzymes, maintaining CCM activity even under high CO 2 conditions, demonstrating its essential role in suppressing the system when carbon is abundant. This finding illuminates how algae balance energy consumption with carbon capture and offers a new target for engineering strains that fix CO 2 more efficiently for biofuel production or climate-mitigation technologies.
    Date: 2025-07-14
    Authors: Shimamura D, Yasuda J, Yamahara Y, Nakano H, Ozawa S, Tokutsu R, Yamagami A, Matsushita T, Takahashi Y, Nakano T, Fukuzawa H, Yamano T.
    Ref: bioRxiv
  12. ABSTRACT Genetically-engineered microbes have the potential to increase efficiency in the bioeconomy by overcoming growth-limiting production stress. Screens of gene perturbation libraries against production stressors can identify high-value engineering targets, but follow-up experiments needed to guard against false positives are slow and resource-intensive. In principle, the use of orthogonal gene perturbation approaches could increase recovery of true positives over false positives because the strengths of one technique compensate for the weaknesses of the other, but, in practice, two parallel screens are rarely performed at the genome-scale. Here, we screen genome-scale CRISPRi (CRISPR interference) knockdown and TnSeq (transposon insertion sequencing) libraries of the bioenergy-relevant Alphaproteobacterium, Zymomonas mobilis , against growth inhibitors commonly found in deconstructed plant material. Integrating data from the two gene perturbation techniques, we established an approach for defining engineering targets with high specificity. This allowed us to identify all known genes in the cytochrome bc 1 and cytochrome c synthesis pathway as potential targets for engineering resistance to phenolic acids under anaerobic conditions, a subset of which we validated using precise gene deletions. Strikingly, this finding is specific to the cytochrome bc 1 and cytochrome c pathway and does not extend to other branches of the electron transport chain. We further show that exposure of Z. mobilis to ferulic acid causes substantial remodeling of the cell envelope proteome, as well as the downregulation of TonB-dependent transporters. Our work provides a generalizable strategy for identifying high-value engineering targets from gene perturbation screens that is broadly applicable. IMPORTANCE Engineering microorganisms to tolerate harsh production conditions stands to increase bioproduct yields of engineered microbes. In this study, we systematically identified Z. mobilis genes that confer resistance or susceptibility to chemical stressors found in deconstructed plant material. We used complementary genetic techniques to cross-validate these genes at scale, providing a widely applicable method for precisely identifying genetic alterations that increase chemical resilience. We discovered genetic modifications that improve anaerobic growth of Z. mobilis in the presence of inhibitory chemicals found in renewable plant-based feedstocks. These results have implications in engineering robust production strains to support efficient and resilient bioproduction. Our methodologies can be broadly applied to understand microbial responses to chemicals across systems, paving the way for developments in biomanufacturing, therapeutics, and agriculture.
    Date: 2025-07-14
    Authors: Eckmann JB, Enright Steinberger AL, Davies M, Whelan E, Myers KS, Robinson ML, Banta AB, Lal PB, Coon JJ, Sato TK, Kiley PJ, Peters JM.
    Ref: bioRxiv
  13. Divergent transcription from bidirectional promoters is frequently observed in eukaryotic genomes, but the biological relevance of divergent RNA transcripts (DT) is unknown. We identified and characterized BDNF-DT , a novel DT gene, and BDNF-AS-DT , a novel readthrough gene, in the locus containing BDNF , a gene with key roles in neuronal development, differentiation, and synaptic plasticity. BDNF-DT is independent from the known BDNF antisense ( BDNF-AS ), and its expression is developmentally regulated and positively correlated with BDNF in human postmortem dorsolateral prefrontal cortex (DLPFC). BDNF-DT and BDNF-AS-DT expression increase after induced depolarization, but the temporal dynamics follow expression of BDNF, suggesting a regulatory role. Moreover, CRISPR-mediated upregulation of BDNF in human neural progenitor cells drives BDNF-DT expression. Finally, BDNF-DT shows higher expression in DLPFC from patients diagnosed with schizophrenia compared to neurotypical controls, and genetically predicted lower expression of the BDNF-AS-DT readthrough transcript is associated with schizophrenia and with the schizophrenia-associated C allele of the rs6265 single-nucleotide polymorphism. These data suggest that BDNF-DT and BDNF-AS-DT contribute to BDNF regulation and schizophrenia risk.
    Date: 2025-07-14
    Authors: Bach SV, Punzi G, Smith NE, Mukherjee S, Shin JH, Chen Q, Pertea G, Collado-Torres L, Maynard KR, Page SC, Kleinman JE, Hyde TM, Weinberger DR, Martinowich K, Ursini G.
    Ref: medRxiv
  14. CRISPR-Cas nucleases have revolutionized diagnostics and biotechnology by providing programmable specificity. Here, we extend the understanding of Cas12a biology with a screen that, unexpectedly, finds that Cas12a trans cleavage activity can be modulated by nicks in the protospacer in a position-dependent manner. Wanting to explore the impact of non-conventional trans cleavage substrates, we subsequently find that non-specific Cas12a cleavage can be significantly reduced with RNA and chimeric (mixed RNA/DNA) reporter sequences. Exploiting these features, we introduce RAPID ( R NA/DNA A dvanced chimeric, P AM-free, I ntegrated Nicking, D iagnostics), a PAM-independent nucleic acid detection platform. By strategically introducing a nick within the spacer region, RAPID expands Cas12a detection to include target RNAs, which can be ligated in situ to create a hybrid protospacer-target with trans cleavage activity matching conventional Cas12a. We then apply RAPID to detect single point mutations in ssDNA and RNA substrates, a challenge for traditional Cas12 and Cas13 systems. In combination with RT-LAMP, RAPID is used for PAM-free RNA detection in clinical samples, achieving sensitivity down to ∼1 aM and 100% concordance with RT-qPCR.
    Date: 2025-07-14
    Authors: Iwe IA, Liu FX, Corsano A, da Silva SJR, Doucet J, Singh S, Lamothe G, Zayani R, Nguyen J, Matthews Q, Vigar JR, Bayat P, Simchi M, Bozovicar K, Charania M, Panfilov S, Li X, Mazzulli T, Tremblay JP, Zhao Y, Green AA, Li Z, Yao S, Pardee K.
    Ref: medRxiv
  15. Arabidopsis encodes ten TREHALOSE-6-PHOSPHATE PHOSPHATASE ( TPP ) genes, homologous to maize RAMOSA3 (I), which controls shoot branching. To explore the roles of the arabidopsis TPPs , we analyzed their expression in shoot apices and found distinct spatial patterns, including TPPI and TPPJ expressed in shoot meristem boundaries, reminiscent of RA3 expression. Single and double TPP mutants lacked dramatic phenotypes, however a CRISPR-Cas9 knockout of all ten TPP genes resulted in increased branching, mirroring ra3 mutants in maize, as well as reduced size and earlier flowering. Expression of GFP-tagged TPPI under its native promoter partially complemented these defects, with protein localization in meristems, vascular tissues and in nuclei. Metabolite profiling revealed higher trehalose 6-phosphate (Tre6P), lower trehalose, and altered sugar and iron-associated metabolites. The mutants also developed chlorosis and grew poorly on low-nutrient media, linked to low iron levels, and reversible with iron supplementation. Consistent with these findings, developmental and iron-responsive genes were up-regulated in the mutants, while photosynthesis-related genes were repressed. Our findings suggest that TPP genes redundantly regulate shoot architecture, sugar metabolism, iron homeostasis and photosynthesis in arabidopsis, and support a role for TPP-mediated Tre6P signaling in coordinating developmental and physiological pathways.
    Date: 2025-07-12
    Authors: Skopelitis T, Swentowsky KW, Goldshmidt A, Feil R, Lunn JE, Jackson D.
    Ref: bioRxiv
  16. The genetic basis underlying non-tuberculous mycobacteria (NTM) pathogenesis remains poorly understood. This gap in knowledge has been partially filled over the years through the generation of novel and efficient genetic tools, including the recently developed CRISPR interference (CRISPRi) technology. Our group recently capitalized on the well-established mycobacteria-optimized dCas9 Sth1 -mediated gene knockdown system to develop a new subset of fluorescence-based CRISPRi vectors that enable simultaneous controlled genetic repression and fluorescence imaging. In this Research Protocol, we use the model organism Mycobacterium smegmatis ( M. smegmatis ) as surrogate for NTM species and provide simple procedures to assess CRISPRi effectiveness. We describe how to evaluate the efficacy of gene-silencing when targeting essential genes but also genes involved in smooth-to-rough envelope transition, a critical feature in NTM pathogenesis. This protocol will have a broad utility for mycobacterial functional genomics and phenotypic assays in NTM species.
    Date: 2025-07-12
    Authors: Point V, Achache W, Laudouze J, Sepulveda Ramos E, Maziero M, Crauste C, Canaan S, Santucci P.
    Ref: bioRxiv
  17. Microcystins are potent cyanotoxins produced by toxigenic cyanobacteria during harmful algal blooms (HABs), posing risks to ecosystems and human health. In this study, we developed a portable RPA-CRISPR/Cas12a biosensing platform for the rapid, on-site detection of the microcystin synthetase E ( mcyE ) gene, a key biomarker for microcystin-producing strains. The developed RPA-CRISPR/Cas12a assays enable detection of the mcyE gene within 50 min, with either fluorescence or lateral flow assay readouts. The fluorescence readouts have an analytical detection limit of 48.4 copies/µL and a dynamic range of 1.2 × 10 2 to 1.2 × 10 7 copies/µL. To enable field deployment, a magnetic bead-based DNA extraction method was integrated, achieving extraction within 1 hour without centrifugation. The complete workflow demonstrated a method LOD of 8.4 × 10 2 cells/mL in spiked lake water. Applicability was validated using non-spiked environmental water samples collected from multiple HAB-affected lakes. Importantly, a systematic matrix effect assessment was conducted for the CRISPR sensing step, evaluating environmental variables such as pH, ions, nutrients, and natural organic matter. This study establishes a practical, sensitive, and selective detection tool for proactive HAB monitoring. The platform’s simplicity, portability, and completeness, from sample pretreatment to signal readout, highlight its potential for real-world environmental biosensing applications.
    Date: 2025-07-12
    Authors: Hasan R, Smith M, Wang S.
    Ref: bioRxiv
  18. Prostate cancer is a major cause of cancer-related deaths among men in Sub-Saharan Africa, where late-stage diagnoses are common due to limited access to affordable and sensitive diagnostic tools. Early detection is essential to improve survival and reduce the disease burden. This review explores the integration of epigenetic biomarkers and CRISPR-Cas12a technology as a transformative approach for early, non-invasive prostate cancer detection in resource-limited settings. Among the many complexities of cancer development, molecular dysregulation plays a remarkable role, and epigenetic modifications such as DNA methylation, histone changes, and non-coding RNA expression have emerged as stable and specific biomarkers with significant potential for the early detection and characterisation of prostate carcinogenesis. However, their low concentration in body fluids presents a detection challenge. CRISPR-Cas12a, known for its high specificity and sensitivity, offers a promising solution. When combined with isothermal amplification and liquid biopsy techniques, it enables rapid, low-cost, and point-of-care diagnostics. This review proposes a low-cost, CRISPR-Cas12a-based diagnostic pipeline for detecting prostate cancer-specific epigenetic markers in liquid biopsies. The implementation of this technology in Sub-Saharan Africa could significantly improve early diagnosis, reduce mortality, and advance health equity.
    Date: 2025-07-11
    Authors: Nguedia NK, Amadi EC, Kintung IF, Ogunlana OO, Chinedu SN.
    Ref: Preprints.org
  19. Abstract Long-Read Sequencing (LRS) technologies offer capabilities for characterizing complex engineered DNA constructs from Golden Gate and barcoded DNA variant assemblies, CRISPR engineered libraries or Multiplexed Assays of Variant Effect (MAVE) experiments. However, the heterogeneity of such molecules, combined with potential structural and length variability, presents analytical challenges. We present SLICER (Sequencing Long-read Identifier of Complex Element Regions), a pipeline for analyzing LRS of such constructs. SLICER dynamically identifies and extracts user-defined barcode/core elements per-read using an anchor-based method, accommodating positional/length variations and aligns these back to reference sequences. If absent, SLICER is capable of de novo reference prediction, a feature that can be insightful to identify unpredicted/aberrant phasing/combinatorial events. When benchmarked, SLICER’s d e novo reference prediction was accurate to within 1% of reference data. SLICER’s dynamic extraction and robust de novo reference capabilities provide an invaluable tool for synthetic and engineered biology applications, enabling comprehensive interrogation of complex barcoded DNA constructs and libraries. SLICER is available at https://github.com/mbassalbioinformatics/SLICER.
    Date: 2025-07-11
    Authors: Bassal M, Suryatenggara J, Liu Y, Tenen D.
    Ref: Research Square
  20. Abstract Innate immunity, traditionally viewed as non-specific, is increasingly recognized for its capacity to regulate microbial communities with precision. In the sea anemone Nematostella vectensis , we uncover a form of selective immunity mediated by nematosomes—motile immune cell clusters that preferentially phagocytose foreign Vibrio isolates while sparing native bacteria. We identify the transcription factor cJUN as essential for this process: CRISPR/Cas9-mediated knockout of cJUN impairs nematosome proliferation, reduces lysosomal activation, and alters microbiome composition by allowing colonization of non-native strains. These results link immune gene function to microbial selectivity and demonstrate that even early-diverging animals exhibit immune discrimination. Our findings challenge the classical dichotomy between innate and adaptive immunity and reveal that immune specificity may be evolutionarily ancient. This work establishes Nematostella as a model for studying microbiome-induced innate immune training and highlights conserved mechanisms that maintain host-microbe homeostasis.
    Date: 2025-07-11
    Authors: Fraune S, Kaya N, Abukhalaf M, Reyes GF, Hentschel U, Tholey A.
    Ref: Research Square
  21. ABSTRACT Understanding the dynamic regulation of signaling pathways requires methods that capture cellular responses in real time. While high-content imaging-based genetic screens have transformed functional genomics, they have remained largely limited to static or binary phenotypes. Here, we present DynaScreen, an imaging-based, pooled CRISPR screening platform that enables high-throughput investigation of dynamic cellular phenotypes at single-cell resolution. By integrating Förster resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) biosensors with photoactivation- based single-cell tagging and pooled CRISPR screening technology, we establish a scalable system to identify genes that regulate the timing, amplitude, and duration of signaling responses. As proof of principle, we applied this approach to the cAMP signaling pathway, a key regulator of cellular physiology. Using a custom guide RNA (gRNA) library, we tracked real-time cAMP dynamics in response to agonist stimulation and identified genes that modulate its basal levels and response kinetics. Cells with aberrant signaling were selectively photoactivated, isolated by fluorescence-activated cell sorting (FACS), and subjected to next-generation sequencing to pinpoint causal genetic perturbations. This strategy successfully uncovered known and novel regulators of cAMP dynamics. In conclusion, the integration of FLIM microscopy, CRISPR technology and open-source software to handle image analysis, automated hit identification and data representation, enables real-time exploration of dynamic phenotypes in a wide range of biological settings.
    Date: 2025-07-11
    Authors: Mukherjee S, van Tooren M, Zanetti G, Sfakianakis D, Kemps D, Klarenbeek J, Kuiken HJ, Lieftink C, van den Broek B, Beijerbergen RL, Jalink K.
    Ref: bioRxiv
  22. Summary Mitochondria contain their own genome, the mitochondrial DNA (mtDNA), which is under strict control of the cell nucleus. mtDNA occurs in many copies in each cell, and mutations often only affect a proportion of them, giving rise to heteroplasmy. mtDNA copy number and heteroplasmy level together shape the cell- and tissue-specific impact of mtDNA mutations, ultimately giving rise to rare mitochondrial and common neurodegenerative diseases. However, little is known about how copy number and heteroplasmy interact within single cells, and how this is regulated by the nuclear genes and pathways that sense and control them. Here we describe MitoPerturb-Seq for CRISPR/Cas9-based high-throughput single-cell interrogation of the impact of nuclear gene perturbation on mtDNA copy number and heteroplasmy. We screened a panel of nuclear mtDNA maintenance genes in cells with heteroplasmic mtDNA mutations. This revealed both common and perturbation-specific aspects of the integrated stress-response to mtDNA depletion, that were only partially mediated by Atf4, and caused cell-cycle stage-independent slowing of cell proliferation. MitoPerturb-Seq thus provides novel experimental insight into disease-relevant mito-nuclear interactions, ultimately informing development of novel therapies targeting cell- and tissue-specific vulnerabilities to mitochondrial dysfunction.
    Date: 2025-07-11
    Authors: Burr SP, Auckland K, Glynos A, Dhawanjewar A, Wei W, Ryall C, Hynes-Allen A, Prater M, Sczaniecka-Clift M, Prudent J, Chinnery PF, van den Ameele J.
    Ref: bioRxiv
  23. Harnessing the precision of CRISPR systems for diagnostics has transformed nucleic acid detection. However, the integration of upstream cellular signals into CRISPR-based circuits remains largely underexplored. Here, we introduce a synthetic transduction platform that directly links endogenous DNA repair activity to CRISPR-Cas12a activation. By coupling base excision repair (BER) events to a programmable DNA-based transducer, our system converts the activity of DNA glycosylases, such as uracil-DNA glycosylase (UDG) and human 8-oxoguanine glycosylase (hOGG1), into a robust fluorescence signal via Cas12a-mediated trans-cleavage. This one-step CRISPR-based assay operates directly in cell lysates, enabling rapid and sensitive readout of enzymatic activity with high specificity. Additionally, it also enables in 15 minutes the throughput screening of novel potential inhibitors with high sensitivity. The modular design allows adaptation to diverse repair enzymes, offering a generalizable strategy for transforming intracellular repair events into programmable outputs. This approach lays the foundation for activity-based molecular diagnostics, synthetic gene circuits responsive to cellular states, and new tools for monitoring DNA repair in real time and drug screening.
    Date: 2025-07-11
    Authors: Bagheri N, Bertucci A, Merlo R, Porchetta A.
    Ref: bioRxiv
  24. Noncoding genetic variants underlie many complex diseases, yet identifying and interpreting their functional impacts remains challenging. Late-onset Alzheimer’s disease (LOAD), a polygenic neurodegenerative disorder, exemplifies this challenge. The disease is strongly associated with noncoding variation, including common variants enriched in microglial enhancers and rare variants that are hypothesized to influence neurodevelopment and synaptic plasticity. These variants often perturb regulatory sequences by disrupting transcription factor (TF) motifs or altering local TF interactions, thereby reshaping gene expression and chromatin accessibility. However, assessing their impact is complicated by the context-dependent functions of regulatory sequences, underscoring the need to systematically examine variant effects across diverse tissues, cell types, and cellular states. Here, we combined in vitro and in vivo massively parallel reporter assays (MPRAs) with interpretable machine-learning models to systematically characterize common and rare variants across myeloid and neural contexts. Parallel profiling of variants in four immune states in vitro and three mouse brain regions in vivo revealed that individual variants can differentially and even oppositely modulate regulatory function depending on cell-type and cell-state contexts. Common variants associated with LOAD tended to exert stronger effects in immune contexts, whereas rare variants showed more pronounced impacts in brain contexts. Interpretable sequence-to-function deep-learning models elucidated how genetic variation leads to cell-type-specific differences in regulatory activity, pinpointing both direct transcription-factor motif disruptions and subtler tuning of motif context. To probe the broader functional consequences of a locus prioritized by our reporter assays and models, we used CRISPR interference to silence an enhancer within the SEC63-OSTM1 locus that harbors four functional rare variants, revealing its gatekeeper role in inflammation and amyloidogenesis. These findings underscore the context-dependent nature of noncoding variant effects in LOAD and provide a generalizable framework for the mechanistic interpretation of risk alleles in complex diseases.
    Date: 2025-07-11
    Authors: Chen Z, Liu Y, Brown AR, Sestili HH, Ramamurthy E, Xiong X, Prokopenko D, Phan BN, Gadey L, Hu P, Tsai L, Bertram L, Hide W, Tanzi RE, Kellis M, Pfenning AR.
    Ref: bioRxiv
  25. Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. We evaluated CasX2 ( Plm Cas12e), a class II CRISPR system derived from Planctomycetes , a non-pathogenic bacterium present in aquatic and terrestrial soils. CasX2 offers several advantages over Streptococcus pyogenes Cas9 ( Sp Cas9) and Staphylococcus aureus Cas9 ( Sa Cas9), including its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and no known pre-existing immunity in humans. A recent study reported that a three amino acid substitution in CasX2 significantly enhanced cleavage activity (1). Therefore, we compared cleavage efficiency and double-stranded break repair characteristics between the native CasX2 and the variant, CasX2 Max , for cleavage of CCR5 , a gene that encodes the CCR5 receptor important for HIV-1 infection. Two CasX2 single guide RNAs (sgRNAs) were designed that flanked the 32 bases deleted in the natural CCR5 Δ32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2 Max using sgRNAs with 20 nt and 23 nt spacer lengths, enabled robust genomic cleavage of CCR5 . Structural modeling indicated that two of the CasX2 Max substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2 Max in cellular gene excision. In sum, CasX2 Max consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.
    Date: 2025-07-11
    Authors: Hodge CA, Donegan NP, Armstrong DA, Hayden MS, Howell AL.
    Ref: bioRxiv
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