ABSTRACT Neuroblastoma is a childhood cancer, arising in the developing sympathetic nervous system. Differentiation therapy with 13-cis-retinoic acid (RA) is routinely given to children with high-risk neuroblastoma in the minimal residual disease setting to prevent relapse, however there is little understanding of which patients benefit from RA therapy. ATRX alterations are identified in 10% of high-risk neuroblastomas and associated with poor outcomes. The commonest type of ATRX alterations in neuroblastoma are in-frame multi-exon deletions, followed by nonsense mutations predicted to result in loss-of-function ( ATRX LoF). We treated paired ATRX wild-type and ATRX LoF neuroblastoma cell lines with RA and show that cells with ATRX LoF fail to upregulate direct RA target genes. Cells with ATRX LoF also show reduced chromatin accessibility at genes involved in differentiation and development following RA treatment. Conversely, neuroblastoma models with in-frame deletions mount a response to RA and show in-vitro sensitivity to RA. Taken together this shows that the mechanism of differentiation in ATRX -altered neuroblastoma depends on the type of ATRX alteration, with implications relating to both oncogenesis and therapeutic response.

ABSTRACT Dravet syndrome (DS) is a severe childhood genetic epilepsy, caused by de novo heterozygous mutations in SCN1A , resulting in a loss-of-function of the voltage-gated sodium ion channel, Nav1.1. Nav1.1 is expressed in the brain, and at a lower level, in the heart. DS manifests in the first year of life. Patients exhibit tonic-clonic seizures, febrile seizures, cognitive decline, developmental delays, ataxia, and sudden unexpected death from epilepsy (SUDEP). We have developed a novel AAV-F mediated CRISPR-Cas-inspired RNA targeting system (CIRTS) preclinical treatment to increase endogenous Scn1a and ameliorate the disease phenotype in a clinically-relevant heterozygous loss-of-function mouse model of DS. We designed novel guide RNAs (gRNAs) to target the long non-coding RNA, (or natural antisense transcript) of Scn1a to increase the expression of Scn1a mRNA in DS mice. We show that intracerebroventricular and intravenous administration of AAV-F-CIRTS-gRNA9 to target the brain and the heart to neonatal Scn1a +/- mice resulted in a significant increase in survival, and a reduction in SUDEP, febrile seizures and seizure duration. These findings provide proof-of-concept evidence that an AAV-F-CIRTS mediated therapy hold promise as a potential treatment for DS.

Type III CRISPR systems detect the presence of RNA from mobile genetic elements (MGE) in prokaryotes, providing antiviral immunity. On activation, the catalytic Cas10 subunit conjugates ATP to form cyclic oligoadenylate (cOA) signalling molecules that activate ancillary e\ectors, providing an immune response. Cellular ring nucleases degrade cOA to reset the system. Here, we describe the structure and mechanism of a new family of ring nucleases, Crn4, associated with type III-D CRISPR systems. The crystal structure of Crn4 reveals a small homodimeric protein with a fold unrelated to any known ring nuclease or, indeed, any known protein structure. Crn4 degrades a range of cOA species to linear oligoadenylates in vitro and ameliorates type III CRISPR immunity in vivo . Phage and plasmids also encode Crn4 orthologues that may function as anti-CRISPRs. These observations expand our understanding of ring nucleases and reveal a new protein fold for cyclic nucleotide recognition.

Radiotherapy is part of the standard-of-care for glioblastoma, yet tumors invariably recur as incurable lesions post-treatment. Recent studies suggest that radiation-induced astrocyte reactivity fosters a tumor-supportive environment, however effective strategies targeting reactive astrocyte phenotypes are lacking. Using a novel image-based assay, we screened over 1,700 small molecule compounds, identifying 29 that inhibit radiation-induced astrocyte reactivity in human astrocytes. Among these, Flunarizine, a calcium-entry blocker approved for migraine treatment, significantly reduced astrocyte reactivity in vitro and in vivo. In a genetically engineered glioblastoma mouse model, combining Flunarizine with radiotherapy markedly improved survival without affecting unirradiated controls, indicating specificity for a radiation-induced phenotype. Mechanistically, Flunarizine inhibited radiation-induced fibrosis in vivo and directly suppressed astrocytic TGF-beta activation in vitro. Notably, Flunarizine treatment had no direct effect on primary glioblastoma cells, emphasizing its microenvironmental specificity. In conclusion, we identified Flunarizine as a promising repurposed compound capable of effectively mitigating radiation-induced astrocyte reactivity and delaying glioblastoma recurrence. This approach offers a viable therapeutic strategy to enhance current glioblastoma treatments. Graphical abstract

ABSTRACT Ultraviolet-B radiation (UV-B) poses a major challenge to all forms of plant life. The liverwort Marchantia polymorpha (Marchantia) serves as a key model organism to study signaling pathways and to infer their evolution throughout the green lineage. Marchantia expresses key components of UV-B signaling, including the photoreceptor UV RESISTANCE LOCUS 8 (MpUVR8), the WD40-repeat protein REPRESSOR OF UV-B PHOTOMORPHOGENESIS (MpRUP), the E3 ubiquitin ligase complex CONSTITUTIVELY PHOTOMORPHOGENIC 1 / SUPPRESSOR OF phyA-105 (MpCOP1/MpSPA), and the transcriptional regulator ELONGATED HYPOCOTYL 5 (MpHY5). Here, we show that MpUVR8 exists as a homodimer in its ground-state in vivo, then monomerizes and accumulates in the nucleus upon UV-B activation. Activated MpUVR8 interacts with MpCOP1, triggering growth inhibition, gene expression changes, biosynthesis of UV-absorbing metabolites, and photoprotection, which overall contributes to UV-B stress tolerance. MpRUP facilitates redimerization of MpUVR8 and Mp rup null mutants show enhanced UV-B photomorphogenesis, indicating that MpRUP efficiently represses MpUVR8 signaling. Unlike the case in Arabidopsis and in contrast to the strong Mp cop1 mutant phenotype, Mp spa mutants develop only a weak constitutive photomorphogenesis phenotype, indicating that COP1 function is more independent of SPA in Marchantia than in Arabidopsis. Interestingly, however, Mp spa is linked with a hyper-responsive UV-B phenotype, suggesting that MpSPA is a negative regulator of MpUVR8 signaling. Our findings demonstrate that core components of UV-B signaling existed in the last common ancestor of extant land plants; however, regulatory interactions have diversified in different lineages since their divergence more than 400 million years ago.

CRISPR interference (CRISPRi) screens have emerged as powerful tools for dissecting gene function, yet their application to genes with multiple promoters, which comprise over 60% of human genes, remains poorly understood. Here, we demonstrate that CRISPR-dCas9-based screens exhibit widespread promoter specificity, with untargeted promoters often showing compensatory upregulation to maintain gene expression. Leveraging this selective targeting of individual promoters within the same gene, we developed isoform-specific single-cell Perturb-Seq to systematically analyse alternative promoter function. Our analysis revealed that alternative promoters in 48.3% of targeted genes drive distinct transcriptional programs. This suggests that promoter selection represents a fundamental mechanism for generating cellular diversity rather than mere transcriptional redundancy. In breast cancer models, this promoter-specific targeting revealed differential effects on drug sensitivity, where distinct estrogen receptor (ESR1) promoters showed opposing influences on tamoxifen response and patient survival. These findings demonstrate the necessity of promoter-level analysis in functional genomics and suggest new strategies for therapeutic intervention through promoter-specific targeting.

Objective This study investigated the effects of endogenous C. elegans U6 promoters on dpy-10 gene editing efficiency. Methods We screened endogenous U6 snRNA genes from the WormBase database and constructed 14 editing plasmids targeting dpy-10 by replacing the U6 r07e5.16 promoter in pSX524 (P eft-3 cas9::tbb-2 terminator::U6 r07e5.16 dpy-10 sgRNA ) through molecular cloning. Following standardized microinjection protocols, we quantified gene editing efficiency and high-efficiency gene editing index based on dpy-10 mutant phenotypes in F1 progeny. Results Fifteen U6 snRNA genes ( r07e5.16, f35c11.9, t20d3.13, k09b11.15, k09b11.16, w05b2.8, c28a5.7, f54c8.9, k09b11.11, k09b11.12, k09b11.14, t20d3.12, f54c8.8, f54c8.10, k09b11.13 ) were identified from WormBase database. Comparative analysis revealed four U6 promoters ( w05b2.8, c28a5.7, f54c8.9 , and k09b11.11 ) significantly enhanced gene editing compared to other U6 promoters, including commonly used U6 r07e5.16 and U6 k09b11.12 promoters in C. elegans community. Notably, the gRNA F+E scaffold showed no improvement over the gRNA scaffold when paired with the optimal U6 w05b2.8 promoter. Conclusion This study identifies superior U6 promoters for C. elegans gene editing and demonstrates the critical role of promoter optimization in CRISPR systems, providing novel insights for technical refinement.

Strengthening high-yield phenotypes while maintaining physiological and genetic stability presents a significant challenge in the improvement of high-yield industrial strains (HIS). Coenzyme Q 10 (CoQ 10 ), a crucial quinone electron carrier in the electron transport chain, is widely used in the prevention and treatment of cardiovascular diseases. In this study, the established HIS Rhodobacter sphaeroides HY01, employed for CoQ 10 production, was engineered to enhance productivity while ensuring strain stability. Comparative omics identified the PrrAB two-component system as an oxygen-responsive regulator that links CoQ 10 biosynthesis to photosynthetic pathways. Mutagenesis of PrrA, guided by AlphaFold3 modeling and fluorescence screening, introduced mutations that led to a 37.5% increase in CoQ 10 production. To address phenotypic reversion due to metabolic burden, genome-scale CRISPR interference (CRISPRi) screening identified key genes involved in DNA repair and stress adaptation. Deletions of these genes generated a stable strain that achieved 3.6 g/L CoQ 10 in a 50- L pilot-scale fed-batch fermentation, surpassing previous reports. This study reveals PrrAB-mediated flux partitioning for redox homeostasis and provides a framework for stabilizing burdened phenotypes in photosynthetic microbes, advancing the sustainable production of redox-active metabolites. Bullet points Identified the PrrAB two-component system as a critical global regulator of CoQ 10 biosynthesis in Rhodobacter sphaeroides . PrrA was evolved through fluorescence-based screening and rational protein engineering, significantly enhancing CoQ 10 biosynthesis in industrial high-yield strain. Genome-scale CRISPRi screening identified genes affecting R. sphaeroides HY01 stability enabling targeted modifications to stabilize high-yield CoQ 10 phenotype. Achieved record 3.6 g/L CoQ 10 yield in 50-L pilot-scale bioreactors enhancing microbial productivity stabilizing high-yield phenotypes advancing strain engineering.

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.

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.

Pest management has entered a new era with the emergence of three innovative antisense technologies: RNA interference (RNAi), contact unmodified antisense DNA biotechnology (CUADb), and CRISPR/Cas systems. These approaches function through sequence-specific nucleic acid duplex formation and guided nuclease activity, offering unprecedented precision for targeted pest control. While RNA-guided systems such as RNAi and CRISPR/Cas were originally discovered in non-insect models as fundamental biological defense mechanisms (primarily against viruses), the DNA-guided CUADb system was first identified in insect pests as a practical pest control tool, with its broader role in ribosomal RNA (rRNA) biogenesis recognized later. These discoveries have revealed an entirely new dimension of gene regulation, with profound implications for sustainable pest management. Although RNAi, CUADb, and CRISPR/Cas share some mechanistic similarities, they differ in their mode of action, specificity, and applicability. No single approach provides a universal solution for all insect pests; instead, each is likely to be most effective against specific pest groups. Moreover, these technologies allow for rapid adaptation of control strategies to overcome target-site resistance, ensuring long-term efficacy. This review summarizes the core functional characteristics, potential applications, and current limitations of each antisense technology, emphasizing their complementary roles in advancing environmentally sustainable pest control. By bridging foundational biological discoveries with applied innovations, this work offers new perspectives on integrating these tools into modern pest management frameworks.

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, we developed iPSC-derived i 3 Neuron (i 3 N) 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 other stem cell models of tauopathy, these i 3 N 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 i 3 N platform recapitulates key features of 4R tauopathy and provides a robust system to identify therapeutic modulators of pathological tau.

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.

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.

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 β-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.

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 independently relevant to phenotypes associated with disorders comorbid with substance use, and if genetic factors influence subsequent outcomes when exposure to psychoactive substances happens at an early age, are questions of interest. We generated a stable mutant line in zebrafish using the CRISPR-Cas9 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 WT 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.

The auxin-inducible degron (AID) system has been widely used to conditionally and dynamically deplete proteins in yeast. In this system, the plant hormone auxin promotes OsTIR1-mediated degradation of proteins carrying an auxin-inducible degron tag. However, “basal” degradation of AID-tagged proteins in the absence of auxin has hampered work with essential proteins due to defective or non-viable strains. A second-generation AID system based on the OsTIR F47G mutant was recently introduced to overcome the limitations of basal degradation in budding yeast. However, it remained unclear to what extent the use of OsTIR F47G eliminates basal degradation and how it impacts auxin-induced degradation. Here, by performing a quantitative characterization of the basal and auxin-induced degradation dynamics using OsTIR1 F74G in budding yeast, we find that basal degradation is still detectable and that it depends on OsTIR1 F74G expression levels. We show that also the auxin-induced degradation rates and auxin-induced steady-state concentrations of AID-tagged proteins depend on OsTIR1 F74G expression levels, in addition to the type of auxin used. Lastly, we showcase how increased basal degradation of AID-tagged functional proteins can impair cell growth and lead to unwanted phenotypes. We anticipate that our findings will guide future applications of the AID system in budding yeast by helping researchers select appropriate OsTIR1 F74G expression levels, particularly in studies focused on precise control of degradation dynamics.

Abstract Many CBASS systems defend against viral infections by depleting cellular NAD+ levels, eventually leading to dormancy or death. This abortive infection strategy is beneficial in stopping fast lytic infections, as cells die before spreading the virus to neighboring cells. However, in chronic viral infections, which often occur in archaea, abortive infection could be detrimental, as the cost of immunity may outweigh that of infection. Here we study an archaeal CBASS system that was expressed in the model organism Haloferax volcanii DS2. We demonstrate that this system protects against a chronically infecting virus, HFPV-1, and eliminates the virus after several passages without killing the host. Moreover, cells that cleared the virus become substantially more resistant to subsequent HFPV-1 infections. Cell death only occurs after extensive incubation with HFPV-1. These findings suggest that CBASS can also be beneficial during non-lytic infections, potentially explaining why such systems are relatively common in archaea.

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.

ABSTRACT 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.

Helper NLRs function as central nodes in plant immune networks. Upon activation, they oligomerize into inflammasome-like resistosomes to initiate immune signaling, yet the dynamics of resistosome assembly remain poorly understood. Here, we show that the virulence effector AVRcap1b from the Irish potato famine pathogen Phytophthora infestans suppresses immune activation by directly engaging oligomerization intermediates of the tomato helper NLR SlNRC3. Cryo-EM structures of SlNRC3 in AVRcap1b-bound and unbound states reveal that AVRcap1b bridges multiple protomers, stabilizing a stalled intermediate and preventing formation of a functional resistosome. Leveraging AVRcap1b as a molecular tool, we also capture an additional SlNRC3 resistosome intermediate showing that assembly proceeds in a stepwise manner from dissociated monomers. These findings uncover a previously unrecognized vulnerability in NLR activation and reveal a pathogen strategy that disrupts immune complex assembly. This work advances mechanistic understanding of resistosome formation and uncovers a previously unrecognized facet of pathogen–plant coevolution.

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.

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.

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.

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.