Summary Anti-CD19 chimeric antigen receptor (CAR)-T therapy induces profound remissions in lupus by depleting B cells, challenging the longstanding view that treatment-resistant disease is sustained by long-lived plasma cells. Additionally, emerging relapses highlight the need to understand how pathogenic autoantibodies arise. Using molecular antibody tagging in mice with human monogenic lupus variants, we reveal that autoantibody-producing cell cohorts are long-lived but plasma cells are short-lived, requiring continuous replenishment from proliferating precursors. The spleen acts as a major plasma cell reservoir, with perivascular localization conserved in mice and lupus patients. Relapse after anti-CD19 CAR-T occurred through newly-generated B cells rather than treatment-resistant clones. Plasma cell depletion by anti-BCMA CAR-T failed to eliminate some precursors that become autoantibody-secreting. These findings demonstrate that continuous B cell-to-plasma cell differentiation, not intrinsic plasma cell longevity, sustains pathogenic antibody responses in lupus, supporting a potential benefit of adjunctive therapies after CAR-T, particularly in genetically predisposed patients.

Nervous system development relies on sequential and coordinated formation of diverse neurons and glia from neural progenitor cells (NPCs). In the spinal cord, NPCs of the pMN domain produce neurons early in development followed by oligodendrocyte precursor cells (OPCs), which subsequently differentiate as oligodendrocytes (OLs), the myelinating glia of the central nervous system, later in development. The mechanisms that specify neural progenitor cells to the OL lineage are not yet well understood. Using zebrafish as an experimental model system, we generated single-cell RNA sequencing and single-nuclei ATAC sequencing data that identified a subpopulation of NPCs, called pre-OPCs, that appeared fated to produce OPCs. pre-OPCs uniquely express several genes that encode transcription factors specific to the OL lineage, including Gsx2, which regulates OPC formation in the mouse forebrain. To investigate Gsx2 function in zebrafish OPC specification, we used CRISPR/Cas9 genome editing to create gsx2 loss-of-function alleles. gsx2 homozygous mutant embryos initiated OPC formation prematurely and produced excess OPCs without altering OL differentiation. Using our single-nuclei multi-omics dataset, we predicted a gene regulatory network centered around gsx2 and identified genes that might be transcriptionally regulated by Gsx2. Taken together, our studies suggest that Gsx2 expression in pre-OPCs contributes to the timing of OPC specification.

SUMMARY Transcriptional regulation of transposons and genes by TRIM28 and 5mC is critical for proper mammalian embryonic development, but the specific roles for these mediators in human embryonic and placental lineage remain unclear. We find that loss of TRIM28 has a limited effect on global transposon expression and instead results in upregulation of genes proximal to TRIM28-bound Long Terminal Repeats (LTRs) in both human trophoblast stem cells (hTSCs) and human embryonic stem cells (hESCs). MER11A elements show especially strong regulatory importance in hTSCs: these elements are bound by both TRIM28 and placental transcription factors and show both heterochromatic and euchromatic features. Some genes are positively regulated by MER11A elements in hTSC basal state, while other MER11A-proximal genes show upregulation only upon TRIM28 deletion. By contrast, loss of DNA methylation in hESCs or hTSCs leads to a global increase in transposon expression. While many genic 5mC targets are shared in hESCs and hTSCs, we also observe evidence that a handful of genes important for somatic development are repressed by 5mC in trophoblast, while a small parallel set of placental genes are repressed by methylation in embryonic tissue. Interestingly, loss of DNMT1 causes hESCs to be rapidly lost from culture in a TP53 and mitotic surveillance checkpoint-dependent manner, while hTSCs show little p53 response to DNMT1 loss or DNA damage generally, instead showing gradual mitotic defects and aneuploidy and slow loss from culture. This discrepancy may explain the higher frequency of karyotypic abnormality found in human placental cells. Together, this study charts the role of TRIM28 and DNA methylation in regulating embryonic and placental transcription and demonstrates divergent p53-dependent responses to genomic instability.

ABSTRACT ARGONAUTE (AGO) proteins associate with small RNAs to form RNA-induced silencing complexes (RISCs). Arabidopsis AGO1 effects post-transcriptional silencing by microRNAs (miRNAs) and small interfering RNAs (siRNAs) and is necessary for siRNA amplification through conversion of RISC target RNAs into double-stranded RNA by the RNA-dependent RNA Polymerase RDR6 and its mandatory cofactors SGS3 and SDE5. Many AGO proteins harbor hydrophobic pockets that interact with tryptophan residues, often surrounded by glycine (GW/WG), in intrinsically disordered regions (IDRs) of RISC cofactors. Here, we show that GW/WG dipeptides in the IDR of SGS3 and the hydrophobic pockets in AGO1 are required for fully functional RDR6-dependent siRNA amplification. We also show that this mechanism requires AGO1-specific structural elements, including positively charged residues surrounding the binding pockets, and a conserved, negatively charged patch in the IDR of SGS3. Thus, the same, conserved protein-protein interaction site is used for different purposes in distinct eukaryotic AGO proteins: the GW/WG-mediated TNRC6-Ago2 interaction is crucial for miRNA-guided silencing in metazoans whereas the GW/WG-mediated SGS3-AGO1 interaction facilitates siRNA amplification via RDR6 in plants.

The Danioninae subfamily of teleost fishes boasts up to four hundred distinct species that have evolved to display a stunning diversity of morphological forms. Here we use newly assembled genome sequences of four laboratory and wild zebrafish strains as well as eleven species of the Danio and Danionella genera to explore their phylogenetic history and the genetic basis of pigment pattern diversification. Phylogenomic analyses uncover extensive introgression and incomplete lineage sorting that have obscured phylogenetic relationships within Danio and corroborate an ancient hybrid origin of zebrafish. Whereas D. rerio inherited ancestral horizontal stripes, relatives repeatedly evolved spots and vertical bars. Interspecific complementation tests reveal functional divergence of the adhesion molecule gene igsf11 and the gap junction gene gja5b between the striped zebrafish and Danio species with divergent patterns. Comparative genomic and transcriptomic analyses suggest that protein and regulatory evolution have accompanied pigment pattern diversification. Our analyses elucidate complex genetic changes underlying the phylogenetic history and morphological diversification in the Danio genus. Resolved phylogenetic relationships, available genome assemblies, transcriptomes, and genetic tractability establish Danio fish species as excellent models for biomedical research in vertebrates.

Analyte detection through aptamer-induced signal generation by CRISPR-Cas enzymes has rapidly emerged as a popular biosensing approach. Here, we investigated the implementability and analytical performance of this setup for the detection of diverse small molecule analytes. We selected nine aptamers from the literature targeting seven analytes and tested a commonly used assay design whereby analyte binding by the aptamer liberates a short complementary DNA strand, which in turn activates Cas12a to generate a fluorescence signal. After extensive optimization, the assay functioned for only two of the seven analytes, and several previously reported results could not be reproduced. While Cas12a fluorescence detection was robust, the low success rate is likely due to aptamers not functioning reliably, underscoring the need for careful aptamer validation. Overall, our study provides a critical assessment of aptamer-Cas12a assay performances and discusses potential strengths, limitations, and pitfalls of this biosensing strategy.

SARS-CoV-2 is a positive-sense RNA virus and was responsible for the devastating COVID-19 pandemic. Although the current disease burden is less severe, there are limited treatment options, significant gaps in knowledge, and a looming threat of the emergence of variants and future pandemics. To address these challenges, we performed genome-wide CRISPR knockout screens in a novel human lung cell line NCI-H23ACE2, as well as in HEK293TACE2 cells, with SARS-CoV-2 Wuhan virus, with the aim of identifying host-dependency factors that could predict effective antivirals. We identified four host-directed drugs, donepezil, dH-ergocristine, trametinib and sorafenib, that could potentially be repurposed to treat coronavirus infections. Three of the drugs inhibited SARS-CoV-2, HCoV-229E, and HCoV-OC43, suggesting they could be used as pan-coronavirus antivirals. We also confirmed that SARS-CoV-2 relies on the NRAS/Raf/MEK/ERK signaling pathway for its replication. Our study highlights the robustness and efficiency of a bilateral approach of gene silencing and antiviral screening to identify host-dependency factors and effective antivirals.

ABSTRACT SauUSI is a dimeric, ATP-dependent Type IV restriction enzyme that protects Staphylococcus aureus by cleaving non-self DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. Using biophysical, single-molecule, and nanopore sequencing methods, we show that 5-methylcytosine recognition first induces ATP-driven unidirectional translocation by one helicase-like subunit, displacing its target recognition domain (TRD). The partner TRD can then bind the liberated modified site, stabilizing a growing DNA loop. On symmetrically methylated DNA, both subunits engage in bidirectional loop translocation. Cleavage is triggered by binding a distal methylated site, by preferred DNA sequences, or upon reaching a DNA end. Interactions with other SauUSI dimers or protein roadblocks affect cleavage site distributions but are not required for nuclease activation. While the first cleavages principally generate blunt-ends or one-nucleotide 3′ overhangs, multiple binding-translocation cycles by individual enzymes ultimately shred the modified non-self DNA, neutralizing its threat.

ABSTRACT Endometrial cancer is one of the main gynecological malignancies worldwide, with an estimated 320, 000 new cases annually. Several studies highlight ARHGAP35 as a significantly mutated gene in these tumors. It encodes for the protein p190RhoGAP-A (p190A), which is a major regulator of the small GTPase family of proteins. ARHGAP5 is a paralog of ARHGAP35 that encodes the protein p190RhoGAP-B (p190B). By analyzing human endometrial cancer samples, we found a co-occurrence of mutations in ARHGAP35 and ARHGAP5 genes and we reported that both are less expressed at the mRNA level in tumoral samples compared to healthy tissues. We were interested in understanding the impact of p190A/B under-expression in endometrial cancer and the relationship between the two paralogs. To do so, we have used CRISPR/Cas9 technology to generate HEC-1-A knockout cells for p190A and p190B. We showed that removal of each paralog led to a similar actin remodeling phenotype with the formation of Cross-Linked Actin Networks (CLANs), dependent on the Rho/ROCK pathway. Moreover, proteomic analysis of p190A and p190B knockout cells highlighted similar affected cell functions. Finally, our study demonstrates a synthetic lethality between p190A and p190B where removal of both paralogs is deleterious in endometrial cancer cells, unveiling a potential actionable vulnerability.

ABSTRACT Newly-made secretory and membrane proteins exit the endoplasmic reticulum (ER) in COPII vesicles that form at specialised ER exit sites. These exit sites are typically near to the early Golgi compartments that receive these vesicles. A key player in the delivery of vesicles to the early Golgi is p115 (USO1), a homodimer with a folded head domain and a coiled-coil tail that is anchored to Golgi membranes. p115 has been shown to capture vesicles and to bind to SNARE proteins to promote membrane fusion. Here we report that the head domain of human p115 can bind directly to Sec16A, a large scaffolding protein that organises ER sites and promotes COPII vesicle formation. Structural prediction and deletion mapping define the region of interaction to a conserved motif in the unstructured N-terminal region of Sec16A, and mutations in p115 that block motif binding reduce the efficiency of secretion. This interaction could potentially allow a subset of p115 molecules to reach across from the early Golgi to the ER exit sites to contribute to the large-scale organisation of the early secretory pathway.

APOE4 is the largest genetic risk factor for late-onset Alzheimer’s disease, but the cellular mechanisms by which APOE variants influence risk of disease remain incompletely understood. We have previously found that APOE4 expression led to the intracellular accumulation of lipid droplets in oligodendrocytes, causing decreased myelination. However, the mechanisms by which APOE4 alters lipid metabolism are not fully understood. Here, we leveraged a genome-wide CRISPR screen and ATAC-sequencing in human induced pluripotent stem cell (iPSC)-derived oligodendrocytes to dissect APOE4’s lipid-associated mechanisms of action. Using these approaches, we identified decreased Wnt signaling, and overactive GSK3b activity, as regulators of lipid droplet accumulation in oligodendrocytes. Genetic and pharmacological inhibition of GSK3b reduced lipid droplets in APOE4 oligodendrocytes, and increased myelination in three-dimensional iPSC-derived brain organoids. Finally, we show that pharmacological inhibition of GSK3b reduces lipid droplets and improves myelination in APOE4;PS19 Tau transgenic mice. Together, our results provide a framework for understanding the mediation of APOE4-related changes to oligodendrocyte lipid metabolism and myelination.

Bioscience encompasses studies on living organisms, their components, and their interactions, with the main aim of translating research into useful applications for medical, clinical, industrial, and environmental uses. The broad field of bioscience has witnessed tremendous growth in research output. This study was designed to evaluate the current trends in bioscience research using bibliometric approaches and develop future policy pointers following the innovative systems framework. Research articles focusing on bioscience published between 1883 and 2024 were sourced from the Scopus database. From the retrieved dataset, relevant articles were systematically screened and included for the analysis. Bibliometrix package, VOSviewer, and Microsoft Excel were used to analyze and visualize the parameters. 8,678 documents involving 30,380 authors from diverse institutions across the globe were published in 3,549 sources, with an annual rise of 4.41%. Exponential growth in bioscience research output was observed after 2007 and remained steady till 2024. The United States and China were leading nations for bioscience research. The most relevant affiliation was the University of California, United States, while the most relevant source was the Brazilian Journal of Medical and Biological Research , in terms of the number of publications. Among the authors, Wang Y had the highest number of documents, while the most impactful author was Zhang J. The most frequent keywords in bioscience research included biological research , genetics , procedures , metabolism , biology , DNA , gene expression , proteins , genomics , and fluorescence . Furthermore, themes such as bioinformatics , microRNA , synthetic biology, CRISPR/Cas9 , deep learning , multi-omics , and big data represented the recent research interests. The bioscience research landscape is defined by the dominance of high-income countries and a shift toward omics-driven and data-intensive approaches. Yet global participation remains uneven, with limited representation from low- and middle-income regions. This study highlights the need for inclusive, globally coordinated strategies that foster equitable access, capacity building, and cross-regional collaboration.

The aggregation of the protein alpha-synuclein (αSyn) is a common feature of multiple neurodegenerative diseases collectively called synucleinopathies, for which the pathobiology is not well understood. The different phenotypic characteristics of the synucleinopathies Parkinson’s disease (PD), Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA) have been proposed to originate from the distinct structures adopted by αSyn in its amyloid forms. Here, using covalent labeling and limited proteolysis coupled to mass spectrometry (LiP-MS) in vitro and in situ within neuronal cells and directly in native patient brain homogenates, we show that pathogenic αSyn from distinct synucleinopathies (PD, DLB and MSA) are structurally different. Further, we found that fibrillar structural differences are associated with different fibril interactomes and neuronal responses. We discovered disease-specific ubiquitination patterns and turnover profiles for pathogenic αSyn species, detected molecular pathways responding specifically to the uptake of different αSyn fibrillar polymorphs, and identified a subset of the involved proteins as candidate direct interactors of αSyn. In particular, components of the Ubiquitin-proteasomal System (UPS), including E3 ubiquitin ligases, chaperones, and Deubiquitinating proteins, showed disease/polymorph-specific interaction patterns, possibly accounting for different resistance of patient-derived αSyn fibrils to degradation. Genetic modulation with CRISPR-based tools showed that members of the UPS degradation pathway (three E3 ligases: UBE3A, TRIM25, HUWE1 and the AAA+ ATPase VCP) reduced αSyn inclusions, in a strain-specific manner. LiP-MS also identified sets of proteins with altered protease susceptibility in postmortem brain homogenates of PD, DLB, and MSA patients. These sets were largely disease-specific and included proteins altered in cells treated with fibrils derived from patients with the matching disease. Our findings provide insight into cellular processes involved in the accumulation and turnover of αSyn pathogenic aggregates in PD, DLB and MSA in a disease specific manner and constitutes a resource of potential novel drug targets in these synucleinopathies.

Background Natriuretic peptides (NPs), encoded by the NPPA and NPPB genes, serve as both diagnostic biomarkers and cardioprotective hormones in heart failure. Their expression is tightly regulated by mechanical load, yet the upstream enhancer mechanisms translating hemodynamic stress into transcriptional activation remain incompletely understood. Methods We investigated the Nppa / Nppb super-enhancer (SE), which resides in a well-insulated topologically associating domain (TAD) and regulates stress-inducible expression of Nppa and Nppb . We applied CRISPR-based enhancer perturbation, chromatin accessibility and histone profiling, chromatin conformation assays, genetic mouse models, human iPSC-derived cardiomyocytes, and paired failing human hearts before and after left ventricular assist device (LVAD) unloading. Results Through integrative epigenomic and genetic analyses, we identified a conserved element, CR9, as a dominant hub enhancer within this SE. In neonatal rat cardiomyocytes, CRISPR-based activation and inhibition established that CR9 was both necessary and sufficient for NP induction, whereas neighboring elements (CR7/CR8) displayed modest activity but synergized with CR9, establishing a hierarchical and cooperative SE architecture. In vivo, CR9 deletion in mice markedly suppressed Nppa / Nppb expression and diminished chromatin accessibility and histone acetylation across adjacent enhancers and promoters, underscoring its structural as well as transcriptional role. Reinsertion of CR9, even in reverse orientation, restored transcription, confirming its orientation-independent activity. CR9 function remained confined within TAD boundaries, supporting a locus-specific regulation. RNAscope revealed spatial proximity between CR9 enhancer RNA and Nppa / Nppb nascent transcripts. Developmental profiling revealed a switch in enhancer usage from CR6/CR7 in the fetal heart to CR9 in adulthood, indicating a transition from developmental to stress-inducible regulation. In human induced pluripotent stem cell–derived cardiomyocytes, deletion of CR9 nearly abolished NPPA / NPPB expression, establishing its essential role in a human cellular context. Most notably, in paired human failing hearts before and after LVAD unloading, chromatin accessibility at CR9 was dynamically reversed, providing the first direct evidence that enhancer states are plastic and therapeutically modifiable in the human myocardium. Conclusions CR9 functions as a stress-inducible hub enhancer that coordinates NP transcription under pathological load. This enhancer exhibits reversible activation in human hearts, underscoring enhancer plasticity as a modifiable regulatory layer mediating stress-responsive transcriptional adaptation in the diseased myocardium. Clinical Perspective What is new? We identified CR9 as a stress-inducible hub enhancer within the Nppa / Nppb super-enhancer that is both necessary and sufficient for natriuretic peptide induction, defining a hierarchical and cooperative regulatory architecture controlling Nppa and Nppb expression. We uncovered a developmental switch in enhancer usage from CR6 in the fetal heart to CR9 in adulthood, revealing how stress-inducible enhancer activity emerges as the heart transitions from developmental to adaptive regulation. Using paired failing human hearts before and after LVAD unloading, we provide the first direct evidence that enhancer states are dynamically reversible in the human myocardium. What are the clinical implications? The reversibility of CR9 activity in human heart failure provides a molecular basis for how natriuretic peptide levels decrease when cardiac function improves with effective therapy, reflecting the underlying enhancer plasticity of the diseasesd myocardium. Enhancer plasticity is an emerging and targetable mechanism in heart failure, raising the possibility that stress-inducible enhancers like CR9 could be engineered to switch on cardioprotective genes when the heart is under stress, offering a new strategy for precision gene therapy.

Entomopathogenic nematodes (EPNs) from the genus Steinernema and Heterorhabditis form mutualistic relationships with symbiotic bacteria from the genus Xenorhabdus and Photorhabdus , respectively. Together, these nematode-bacterium pairs infect and kill insect hosts—primarily larvae from the orders Lepidoptera and Coleoptera . This tripartite interaction provides a powerful model for investigating the molecular mechanisms underlying mutualism and parasitism. A key step toward this goal is the development of a genetically tractable EPN. While RNAi has been applied in some EPN species, stable, transgenerational genetic tools remain limited. Here, we establish a robust CRISPR-Cas9 system in the emerging model Steinernema hermaphroditum , a species that is easily cultivated in both in vivo and in vitro conditions and amenable to gonadal microinjection. Notably, its hermaphroditic reproduction simplifies the generation of genetically stable mutant lines. We present a detailed protocol for efficient, targeted gene knockout via microinjection in S. hermaphroditum . As a proof-of-concept, we knocked out a conserved homologue, unc-22 , which causes a twitching phenotype. The CRISPR-Cas9 based genome editing in S. hermaphroditum has potential to be used to express transgene, or to be adapted to other EPN species that are applicable to benefit agriculture.

Centrosome duplication must be tightly regulated to maintain genomic stability. In Caenorhabditis elegans, the APC/C and co-activator FZR-1 function as negative regulators of centrosome duplication by targeting specific substrates for proteolytic degradation. While C. elegans SAS-5 and ZYG-1 have been identified as substrates of APC/CFZR-1, the mechanism by which APC/CFZR-1-dependent degradation influences centrosome assembly remains unclear. Here, we identified SPD-2, the conserved homolog of human CEP192, as a substrate of APC/CFZR-1. We show that loss of APC/CFZR-1 increases both cellular and centrosomal SPD-2 levels, and that SPD-2 physically associates with FZR-1 in vivo. Functional analyses of canonical D-box motifs reveal that D-box1, D-box2, and D-box3 each contribute to SPD-2 degradation, each with different functional consequences. Mutation of D-box3 alone partially rescued zyg-1 mutant phenotypes by restoring centrosome duplication and embryonic viability through increased centrosomal SPD-2 and ZYG-1. In contrast, mutating D-box1 or D-box2 elevated cellular SPD-2 but did not rescue zyg-1, with the D-box1 mutation further reducing centrosomal SPD-2 and exacerbating duplication defects and lethality in zyg-1 mutants. Our results reveal a conserved mechanism for APC/CFZR-1-dependent degradation of SPD-2 and show that its degron motifs have dual functions in degradation and centrosomal localization, ensuring robust control of centrosome assembly during C. elegans embryogenesis.

The intestinal mucus layer consists of secreted and transmembrane (TM) mucins expressed on the apical surface of enterocytes. The TM mucin MUC1 has a highly O-glycosylated extracellular domain (ED) and a cytoplasmic tail (CT) with signaling potential. MUC1 is a target for the Salmonella adhesin SiiE, which mediates apical invasion of the bacterium into enterocytes. Here, we determined the contributions of the MUC1 ED and CT to Salmonella invasion and subsequent host immune responses. Enzymatic removal of the MUC1 ED from HT29-MTX intestinal cultures blocked Salmonella invasion to levels comparable to MUC1 knockout cells. CRISPR-mediated targeted deletion of the MUC1 CT (MUC1-ΔCT) did not quantitatively affect Salmonella invasion. To investigate downstream host responses, RNAseq transcriptomics analysis of uninfected and Salmonella -infected MUC1-WT, MUC1-ΔCT, and ΔMUC1 cultures was performed. Deletion of full-length MUC1 greatly altered the transcriptome, while only a small group of 132 genes was differentially expressed in MUC1-ΔCT cultures during infection. Several of these CT-dependent genes are related to the NFκB pathway. Immunoblot analysis demonstrates that under uninfected conditions, expression of NFκB subunits RelB, NfkB1-p105, NfkB2-p100, and IκBα was significantly lower in MUC1-WT compared to MUC1-ΔCT and ΔMUC1 cultures. Secretion of cytokines and immune factors was severely reduced in ΔMUC1 cultures, coinciding with reduced Salmonella invasion. In MUC1-ΔCT cultures, only galectin-3 and IL-18 secretion were significantly reduced. We conclude that the MUC1 ED is essential for Salmonella invasion, while the CT modulates the canonical and non-canonical NFκB pathway, pointing at distinct roles for MUC1 domains in microbe-host interactions and signaling. Importance The intestinal mucus layer plays an important role in separating commensal and pathogenic microbes from the underlying epithelium. The transmembrane mucin MUC1 is expressed by different types of intestinal epithelial cells and is thought to have important protective and signaling functions. However, enteropathogenic Salmonella bacteria can hijack MUC1 through engagement with the SiiE adhesin which leads to bacterial invasion of enterocytes at the apical surface. In this study, we determined how the different MUC1 domains contributed to Salmonella invasion and subsequent host responses. We found that the glycosylated MUC1 extracellular domain, but not the cytoplasmic tail, is essential for bacterial invasion. In infected and uninfected intestinal cultures, the MUC1 cytoplasmic tail modulates immune responses including NFκB activation and cytokine secretion. Our study contributes to our understanding of the diverse functions of transmembrane mucins at the intestinal microbe-host interface.

Jasmonic acid (JA) is the precursor of the bioactive molecule jasmonoyl-isoleucine (JA-Ile), a plant hormone that regulates fitness and development. Although JA biosynthesis, signaling, and responses have been intensively studied, the catabolism of JA remains incompletely understood. Here, we used the recently developed technique of limited proteolysis-coupled mass spectrometry (LiP-MS) to investigate metabolite–protein interactions in plants, aiming to discover enzymes involved in JA metabolism. We identified several previously reported JA-binding proteins, thus validating the robustness of the method, along with recognized enzymes of the JA pathway and a series of novel potential JA-binding proteins. We performed functional characterization of a set of identified JA-interacting UDP-glucuronosyltransferase (UGT) enzymes through omics, biochemical, enzymatic, and structural analyses. Our results demonstrate that two tomato UGTs effectively glucosylate JA to form JA-glucosyl esters, potentially playing a role in the regulation of bioactive JA homeostasis. With this, our findings uncovered a missing step in the metabolism of JA.

ABSTRACT Heterotaxy (HTX) syndrome is a congenital disorder characterized by abnormal left-right organ placement, often leading to severe congenital heart defects (CHD). Despite advances in sequencing, many CHD/HTX-associated genes remain functionally unvalidated, hindering effective clinical diagnosis and management. Here, we leveraged a high-throughput CRISPR/Cas9 screening approach in the Xenopus model to rapidly evaluate candidate genes identified from whole-exome sequencing of human CHD patients. Our screen identified Filamin B (FLNB), an actin-binding protein previously linked to skeletal disorders but not to ciliopathies or CHD. We identified 5 probands with CHD/HTX, 3 with recessive, and 2 with damaging heterozygous mutations in FLNB. Disrupting FLNB in Xenopus reproduced key features of the human HTX phenotype, including defects in cardiac development and impaired motile cilia function. Rescue experiments confirmed the functional conservation of human FLNB, directly implicating actin cytoskeletal disruption in ciliogenesis and left-right patterning defects. Our results provide crucial evidence linking human FLNB dysfunction to ciliopathies and CHD/HTX.

CRISPR-based genetic perturbation screens paired with single-cell transcriptomic readouts (Perturb-seq) offer a powerful tool for interrogating biological systems. Yet the resulting datasets are heterogeneous—particularly in vivo —and currently used cell-level perturbation labels reflect only CRISPR guide RNA exposure rather than perturbation state; further, many perturbations have a minimal effect on gene expression. For perturbations that do alter the transcriptomic state of cells, intracellular guide RNA abundance exhibits a dose-response association with perturbation efficacy. We combine (i) per-perturbation, expression-only classifiers trained with non-negative negative–unlabeled (nnNU) risk to yield calibrated scores reflecting the perturbation state of single cells and (ii) a monotone guide abundance prior to yield a per-cell pseudo-posterior that supports both assignment of perturbation probability and selection of affected gene features. To obtain a low-dimensional representation that allows for the accurate reconstruction of gene-level marginals for counterfactual decoding, we train an autoencoder with a quantile–hurdle reconstruction loss and feature-weighted emphasis on perturbation-affected genes. The result is a perturbation-aware latent embedding amenable to downstream trajectory modeling (e.g., optimal transport or flow matching) and a principled probability of perturbation for each non-control cell derived jointly from its guide counts and transcriptome.

Basal breast cancer subtype is enriched for triple-negative breast cancer (TNBC) and exhibits a recurrent large chromosomal deletion in chromosome 4p (chr4p). Chr4p loss is associated with poor survival, evolves early in tumorigenesis and confers on cells a proliferative state. Here, we map the integrated metabolic complex genetic interaction network of chr4p in basal breast cancer to identify targetable vulnerabilities. Differential gene expression analysis of patient derived xenografts and cancer cell models revealed that chr4p loss is associated with changes in cellular energetics and reduction/oxidation balance. Analysis of DepMap pooled genome-wide CRISPR-Cas9 screens identified complex genetic interactions specific to chr4p deletion in basal breast cancer cell models. Functional assays revealed that chr4p loss is associated with disrupted mitochondrial respiratory function and reduced glycolytic capacity, suggesting metabolic rewiring. Increased reactive oxygen species and lipid peroxidation compromised antioxidant defense mechanisms. Ultimately, this study sheds light on targeted therapies for basal breast cancer harboring large chromosomal deletions.

ABSTRACT CRISPR technologies has become an integral part of plant biotechnology, synthetic biology and basic plant research, routinely used by researchers for targeted genome modifications. CRISPR guide RNAs (gRNAs) undermines the highly programmable nature of CRISPR, enabling site-specific genome editing. However, different gRNA targets showed highly variable on-target effectiveness and poor gRNA design could amount to wasting valuable scientific resources. There has been broad development of computational and web-based tools for gRNA efficiency predictions but their performances in plant genome editing remains controversial or untested. Hence, in this study, we systematically evaluated over 20 accessible, web-based in silico gRNA on-target efficiency prediction tools using an experimental plant genome editing dataset. Excitingly, we identified multiple tools, mostly developed using machine learning, that were highly predictive of gRNA on-target genome editing efficiency in planta . The prediction scores assigned to gRNAs in the dataset by these tools were significantly correlated with the frequency of CRISPR-mediated InDels in plants. Furthermore, we evaluated efficiency prediction scores available on popular platforms such as CRISPOR and CRISPR-P which contain large numbers of non-model plant genomes. Our analysis showed that some prediction scores on CRISPOR performed quite well which allows efficient integration of on-target and off-target predictions. Overall, we believe that our study provided insights on improving gRNA design during conventional plant genome editing workflows and should also help unfamiliar researchers interested in CRISPR/SpCas9 genome editing.

ABSTRACT BACKGROUND Genetic susceptibility is a major determinant in intracranial aneurysm (IA) formation and rupture, yet the underlying mechanisms linking genetic variation to vascular dysfunction remain largely unknown. We have identified mutations in ARHGEF17, a guanine nucleotide exchange factor that regulates RhoA activation and cytoskeletal organization, as potential risk variants for IA. Given ARHGEF17’s regulatory role in endothelial barrier integrity and actin remodeling, we hypothesized that ARHGEF17 deficiency promotes IA pathogenesis through dysregulation of the RhoA/ROCK2/MLC signaling axis, leading to endothelial dysfunction and vascular wall instability. METHODS CRISPR–Cas9–mediated ARHGEF17 knockout (ARHGEF17 ⁻/⁻ ) mice and morpholino-based ARHGEF17-deficient zebrafish were established to assess the in vivo vascular effects of ARHGEF17 loss. An intracranial aneurysm model combining elastase injection and deoxycorticosterone acetate (DOCA)–induced hypertension was used to evaluate aneurysm incidence, rupture rate, and survival. Structural remodeling of the Circle of Willis (CoW) was assessed by Victoria Blue, EVG, and Picrosirius Red staining, as well as immunofluorescence for α-SMA, OPN, CD31, and inflammatory markers. Complementary in vitro studies were performed in HUVECs using lentiviral ARHGEF17 silencing (three shRNAs of varying efficiency) to examine endothelial proliferation, migration, tube formation, and barrier function (TEER). Activation of RhoA/ROCK2/MLC signaling was quantified by G-LISA and Western blotting. The ROCK inhibitor Y-27632 (10 μM) was applied to determine pathway dependence. RESULTS ARHGEF17 ⁻/⁻ mice exhibited a significantly higher incidence and rupture rate of intracranial aneurysms, accompanied by fragmentation of elastic fibers, loss of collagen organization, vascular smooth muscle cell dedifferentiation, and robust inflammatory activation in the CoW. Zebrafish lacking ARHGEF17 showed frequent intracranial hemorrhage and compromised vascular wall integrity, further confirming ARHGEF17’s role in cerebrovascular stability. In ECs, ARHGEF17 knockdown impaired proliferation, migration, tube formation, and barrier integrity in a silencing-efficiency–dependent manner. Mechanistically, ARHGEF17 deficiency activated the RhoA/ROCK2/MLC pathway, leading to increased phosphorylation of MLC and MYPT1 and disorganization of F-actin and junctional proteins. Pharmacological inhibition with Y-27632 restored endothelial function, normalized cytoskeletal structure, and re-established junctional continuity, indicating a ROCK2-dependent mechanism. CONCLUSIONS Our findings establish ARHGEF17 as a critical regulator of cerebrovascular integrity and identify RhoA/ROCK2/MLC mediated cytoskeletal remodeling as the mechanistic link between ARHGEF17 deficiency and aneurysm pathogenesis. Loss of ARHGEF17 compromises endothelial barrier function, triggers vascular inflammation, and promotes aneurysm formation and rupture. Importantly, ROCK inhibition rescues endothelial dysfunction, highlighting the RhoA/ROCK2/MLC axis as a promising therapeutic target for ARHGEF17 mutation–associated intracranial aneurysms.

Anopheles gambiae is one of the principal vectors of human malaria. Over the past two decades, transgenic mosquito strains have been essential tools for studying mosquito biology and developing genetic control strategies such as gene drives. Mosquito transformants are typically identified using fluorescent markers, which are assumed to be phenotypically neutral. While generating CRISPR-based gene drive strains carrying an OpIE2 -DsRed marker we unexpectedly found that transgenic females were unable to blood-feed and were consequently sterile, whereas males initially appeared normal and fertile. Given the potential utility of dominant, female-specific sterility for mosquito control, we established additional strains controlling for transgene content and integration site, confirming that the OpIE2 -DsRed cassette caused the defect. Behavioral assays showed that females exhibited normal attraction to a membrane feeder but failed to initiate blood-feeding, performing repeated cycles of probing and proboscis grooming in rapid succession before ultimately leaving the feeder unfed. Microscopy showed that both sexes possessed a distally curved proboscis, providing a morphological explanation for the blood-feeding defect of females and the reduced male lifespan. A second promoter variant ( OpIE2b ), differing in flanking sequences at the IE-2 junction, drove strong marker expression without impairing blood-feeding or longevity. These findings demonstrate that minor differences in promoter architecture can produce major, unexpected phenotypic effects. OpIE2b provides a robust, phenotypically neutral marker for An. gambiae research, while OpIE2a highlights the need for rigorous validation of transgenic components intended for research and applied releases.

Ganoderic Acid DM (GA-DM), a triterpenoid derived from Ganoderma lucidum , exhibits anti-cancer and anti-diabetic activities, but the underlying mechanisms of action remain unclear. To identify genetic modulators of GA-DM response, we conducted a genome-wide CRISPR/Cas9 knockout screen in human melanoma cells. The screen revealed key roles for genes regulating lipid metabolism and inflammatory signalling, particularly the SREBP (Sterol Regulatory Element-binding Protein) and NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) pathways, in the cellular response to GA-DM. While loss of genes involved in regulation of cholesterol biosynthesis conferred resistance to GA-DM, the disruption of ubiquitin-mediated proteolysis and the Hippo pathway genes sensitised cells to GA-DM. Inflammatory genes enriched at later time points suggests that a delayed cellular response contributes to cytotoxicity. Our findings propose a mechanistic model wherein GA-DM perturbs lipid and inflammatory pathways to exert cytotoxic effects and highlights potential targets to enhance its therapeutic efficacy. This work demonstrates the utility of functional genomics in elucidating natural product mechanisms and guiding rational drug development.