FACS-based CRISPR screening has emerged as a potent tool for dissecting the genetic networks that regulate cell-surface glycosylation. However, existing protocols are tedious and poorly suited to many cell models. We developed a lectin-based magnetic-activated cell sorting platform (Lec-MACS) that enables rapid identification of genes controlling expression of specific cell-surface glycans. Lec-MACS is faster and easier to perform than FACS-based screening while producing data of similar quality. We subsequently applied Lec-MACS to produce a genomic atlas of genes regulating breast cancer hypersialylation. This method will dramatically expand the scope and throughput of genetic screens targeted at cell-surface glycans.

Tephritidae insect pests account for extensive crop damage and yield losses globally. Modern, sustainable pest management approaches are species-specific and, therefore, high-quality genome assemblies are required for their application. Here, we present chromosome-level assemblies for five members of the Tephritidae family: Anastrepha fraterculus, Anastrepha ludens, Bactrocera dorsalis, Bactrocera zonata and Zeugodacus cucurbitae . The assemblies used long read sequencing polished with short read sequencing and scaffolded using Hi-C (chromatin conformation capture) sequencing. Prior to scaffolding the assembly deduplication was performed to separate a primary assembly and an alternate assembly, and each was then scaffolded independently. The scaffolded assemblies reached N50 length in the range of 60Mb to 120Mb. The scaffolded assemblies were verified with BUSCO and completeness was in the range 97% to 98.5% and had very low duplicated, fragmented and missing orthologs.

Background and Aims Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy characterized by diagnosis at advanced stages, limited therapeutic options, and frequent resistance to therapies. Although oncogenic KRAS mutations are central drivers of PDAC, alternative pathways indisputably contribute to its tumorigenesis and progression. RhoV, a member of the Rho family of small GTPases, has been implicated in tumor development in other cancer types, such as breast cancer and lung adenocarcinoma; however, its role in PDAC remains unclear. Methods In this study, we investigated the expression and functional impact of RhoV on PDAC. Analysis of publicly available datasets and immunohistochemical profiling of 114 PDAC patient specimens were used to evaluate the expression of RhoV in PDAC and its prognostic impact. Overexpression and CRISPR-Cas9-mediated knockout of RhoV were established in three pancreatic cancer cell lines. Functional analyses, such as cell proliferation, migration, invasion, colony formation, spheroid growth, and mouse xenograft, were used to evaluate the role of RhoV in PDAC cells. Results RhoV overexpression was associated with reduced overall and recurrence-free survival in public datasets and our own patient cohort. Functional assays demonstrated that RhoV overexpression promoted PDAC cell proliferation, colony formation, and spheroid growth, whereas knockout of RhoV suppressed these changes. Moreover, RhoV enhanced PDAC cell migration and invasion in vitro , accompanied by downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating induction of epithelial–mesenchymal transition. Mechanistically, RhoV overexpression activated key MAPK pathway components, including phosphorylation of ERK, JNK, and p38. In vivo , xenograft models confirmed that RhoV drives tumor growth and increases tumor burden. Conclusion These results establish RhoV as a novel oncogenic factor in PDAC progression and highlight its potential as a biomarker and therapeutic target, warranting further investigation into combinatorial targeting strategies to overcome KRAS inhibitor resistance.

Background Musculoskeletal diseases are a leading cause of global disability and healthcare burden, yet traditional orthopaedic procedures often fail to address molecular drivers of degenerative and genetic conditions. CRISPR-Cas Systems, a precise and programmable genome-editing technology, show promise in preclinical musculoskeletal models, ranging from gene knockouts in osteoarthritis to mutation correction in skeletal dysplasia. Despite growing interest, no comprehensive synthesis exists to map how CRISPR-Cas Systems are being applied in orthopaedic research. This scoping review aims to fill that gap. Methods This protocol will be registered with the Open Science Framework and follows PRISMA-ScR guidelines and the Arksey & O’Malley framework. MEDLINE, Embase, Scopus, Web of Science, and grey literature sources from 2005 onward will be searched. Inclusion criteria encompass original research using CRISPR-Cas Systems in human, animal, or in vitro musculoskeletal models. Two reviewers will independently screen titles, abstracts, and full texts using Covidence software. Data extraction will be standardized and performed in duplicate. Extracted variables include study design, model system, target tissue, gene-editing approach, delivery system, and reported outcomes. Results will be synthesized descriptively and thematically. Expected Results We anticipate mapping the evolution and diversity of CRISPR-Cas Systems applications across musculoskeletal tissues (bone, cartilage, tendon, muscle), highlighting domains such as tissue regeneration, gene correction, and disease modeling. Delivery strategies (viral vectors, nanoparticles) and translational challenges, including off-target effects and delivery barriers, will be summarized. Conclusions This will be the first scoping review to systematically characterize the role of CRISPR-Cas Systems in musculoskeletal medicine. Findings will inform researchers, clinicians, and policymakers, helping to guide future translational research and accelerating the integration of gene editing into clinical practice.

Advances in functional genomic technology, notably CRISPR using Cas9 or Cas12, now allow for large-scale double perturbation screens in which pairs of genes are inactivated, allowing for the experimental detection of genetic interactions (GIs). However, as it is not possible to validate GIs in high-throughput, there is no gold standard dataset where true interactions are known. Hence, we constructed a Double-CRISPR Knockout Simulation (DKOsim), which allows users to reproducibly generate synthetic simulation data where the single gene fitness effect of each gene and the interaction of each gene pair can be specified by the investigator. We adapted Monte-Carlo randomization methods to extend single knockout simulation methods to double knockout designs, which simulate the gene-gene interactions between all possible combinations of the input genes. Using DKOsim, we generated simulated datasets that closely resemble real double knockout CRISPR datasets in terms of Log Fold Change (LFC), GI distribution, and replicate correlation. We further inferred optimal CRISPR library designs by systematically investigating critical experimental parameters including depth of coverage, guide efficiency, and the variance of initial guide distribution. This simulation scheme will help to identify optimal computational methods for GI detection and aid in the design of future dual knockout CRISPR screens. Author Summary We designed DKOsim to simulate CRISPR double knockout screens by modeling cell division behavior with both single knockout (SKO) and double knockout (DKO) constructs via Monte-Carlo randomization samplers. Running DKOsim at large scale, we identified the asymptotic tuning points that optimize genetic interaction (GI) identification performance by the delta-LFC (dLFC) method compared to the simulated truth. We show that DKOsim is tunable to approximate actual dual-CRISPR knockout screening data. Comparing replicate correlation from DKOsim with experimentally generated data, DKOsim can be tuned based on users’ desires to reproduce a similar level of randomness to that observed in variety CRISPR screening conditions.

ABSTRACT Insufficient protein intake, leading to malnutrition, is a major global health concern that compromises the immune system and increases susceptibility to diseases. In scenarios where protein availability is constrained, organisms may experience strong selection to efficiently allocate their resources between immunity vs other energy-demanding processes, such as reproduction, resulting in evolutionary trade-offs. Additionally, in many species, protein deficiency has a more significant impact on female reproduction than on males, potentially leading to pronounced sexually dimorphic trade-offs involving immunity and infection outcomes. However, there are no experiments to test these possibilities. In this work, we demonstrate that in Drosophila melanogaster populations selected for increased early-life reproduction under protein limitations, evolved virgin females indeed suffered a greater reduction in their resistance to the pathogen Providencia rettgeri and showed lower survival following infection than males, corroborating our expectations. However, mating resulted in a loss of sexually dimorphic infection outcomes, causing both sexes to exhibit nearly identical infection costs and reduced infection tolerance compared to their ancestral, unselected populations. Moreover, several immune components, including the Toll- and IMD-mediated inducible immune pathways, were either less upregulated or more downregulated in the selected flies, which may contribute to their heightened susceptibility to pathogens. Downregulation in key metabolic pathways and genes related to phagocytosis, melanisation and ROS-mediated defence after infection in selected flies can also be associated with their increased pathogen vulnerability. Taken together, our work thus reveals the reproductive status- and sex-specific plasticity of immune investments and post–infection health in response to evolutionary constraints under chronic protein deprivation.

The CRISPR-Cas system enables precise genome engineering of cell therapies. For allogeneic applications, multiplex editing is frequently required to improve efficacy, persistence, and safety. However, strategies involving multiple DNA double-strand breaks (DSBs) induce genotoxicity by provoking chromosomal aberrations. Base editors, which enable sequence changes without generating DSBs, are widely used for gene disruption, but their capacity for gene insertion remains unexplored. Here, we developed B ase e ditor-mediated k nock- i n ( BEKI ), a non-viral platform that allows targeted transgene insertion in parallel with multiplex gene disruption using a single base editor. Repurposing the Cas9 nickase domain of base editors generates paired nicks, inducing homology-directed repair (HDR). In human T cells, optimized guide RNA orientation and nick distance, together with HDR-enhancing modulators, enabled efficient transgene knock-in at the TRAC , CD3ζ, B2M, and CD3ε loci. Simultaneous base editing of multiple additional genes produced chimeric antigen receptor (CAR) T cells with increased cytokine secretion, drug resistance, and resistance to allo-rejection. Compared to multiplex editing with Cas9, BEKI markedly reduced chromosomal translocations. BEKI therefore provides a streamlined, scalable strategy for multiplex CAR T-cell engineering with a single enzyme, offering a safer route to clinical-grade manufacturing of off-the-shelf therapies for cancer and autoimmune diseases. Graphical Abstract

Abstract Acute Myeloid Leukemia (AML) remains challenging to treat, especially in cases with mutations in the BCL-6 co-repressor (BCOR), which are associated with poor prognosis and chemo-resistance. In this study, we reveal a synthetic lethal interaction between BCOR and dihydroorotate dehydrogenase (DHODH). We demonstrate that BCOR -deficient cells have a heightened sensitivity to DHODH inhibitors such as brequinar and leflunomide, that are already in clinical use. We confirm that DHODH inhibition selectively induces cell death in BCOR-mutant cells in multiple cellular models, in malignant and non-malignant cells, through chemical and genetic manipulation. Interestingly, we find that the dependency on DHODH does not stem from its role in de novo pyrimidine biosynthesis disruption. Rather, DHODH’s role in the electron transport chain, essential for mitigating reactive oxygen species, may be the physiological vulnerability that pushes BCOR-mutant cells toward cell death when DHODH is inhibited. DHODH inhibitors could be repurposed as targeted therapies for BCOR-mutant tumors, offering a promising strategy for precision medicine in AML and other cancers.

Doxorubicin (DOX) is an effective anticancer therapeutic but exhibits dose-dependent, potentially life-threatening cardiotoxicity. The specific mechanisms driving this cardiotoxicity are not fully understood but can include the induction of oxidative stress and subsequent cell death mechanism activation. This has prompted the exploration of NRF2, a master co-ordinator of antioxidant and largely cytoprotective pathways, as a potential approach for the alleviation of DOX-induced cardiotoxicity. Here, NRF2 was pharmacologically activated via CDDO-Me (hitherto referred to as CDDO) to reduce the negative consequences on AC16 human cardiomyocyte cell health and functions. NRF2 intracellular dynamics were quantitatively measured using live-cell imaging, demonstrating rapid (∼10 min) yet sustained (≥24 h) induction of NRF2 expression and functional downstream activity. Genetic perturbations of the NRF2-KEAP1 system highlight that CDDO acts specifically through NRF2 to exert AC16 cytoprotection from DOX whilst not promoting human lung and pancreatic cancer cell line viability. Via RNA-seq analysis, we reveal that CDDO dampens DOX-mediated effects on p53 signalling, apoptosis and ferroptosis. This study provides novel insight into NRF2 dynamics in the widely utilised AC16 cells whilst further elucidating the molecular mechanisms contributing to DOX cardiotoxicity and potential NRF2-orchestrated defence. Graphical abstract

Cerebral ischemic small vessel disease (SVD) is the leading cause of vascular dementia and a major contributor to stroke. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic form of familial SVD. CADASIL is caused by dominant missense mutations in Notch3, a receptor expressed in mural cells, including smooth muscle cells (SMCs) and pericytes. However, the cell-type specific contributions driving the CADASIL pathology remain unknown due to lack of animal models. Here, we generated two conditional knock-in mouse models carrying the CADASIL-causing Notch3 R170C mutation selectively in SMC and brain pericytes. Both Notch3 R 170 C models showed perivascular accumulation of the NOTCH3 extracellular domain, yet developed distinct neurovascular changes depending on the affected cell type. Pericyte-specific Notch3 R170C mice displayed pronounced region-selective microglial activation and vascular changes, whereas SMC-specific Notch3 R170C mice showed localized perivascular gliosis with minimal vascular remodeling. Proteomic profiling of isolated brain vessels revealed largely unique cell-specific responses. Pericytes Notch3 R170C expression dysregulated metabolic pathways, whereas SMC Notch3 R170C expression induced immune signaling related pathways. Integration with single-cell RNA-seq data revealed that many of the proteomic and phosphoproteomic shifts might also include brain endothelial cells, including metabolic changes in the presence of pericyte Notch3 R170C and inflammatory signaling in the presence of SMC-Notch3 R170C . Together, these findings define mural cell-specific mechanisms that contribute to the CADASIL-associated vascular pathology.

Modeling human epithelial diseases and developing cell-based therapies require robust methods to expand and manipulate epithelial stem and progenitor cells in vitro . Basal stem/progenitor cells from stratified epithelia can be expanded in 3T3-J2 fibroblast feeder cell co-culture systems, and the addition of the ROCK inhibitor Y-27632 enhances proliferation and culture longevity, a phenomenon described as ‘conditional reprogramming’. Here, we present a method incorporating the small molecule WS6 to further improve the proliferation and lifespan of cultured epithelial cells from multiple tissues, including airway, skin, and thymus. Cells maintained in this medium (‘EpMED’; FAD+Y+WS6) retain basal stem/progenitor cell identity and function, including the capacity to differentiate. We demonstrate their capacity to engraft in vivo in a tracheal transplantation model. In a second application, we generate clonal CRISPR-Cas9 genome edited nasal cultures, introducing targeted knockouts of DNAH5 or DNAI2 to create primary ciliary dyskinesia disease models. We anticipate that our method will have broad applications in epithelial cell biology, disease modeling, and regenerative medicine, while reducing reliance on immortalized or cancer cell lines and animal experimentation.

Colorectal carcinoma (CRC) remains a major cause of cancer-related mortality, with rising incidence in individuals under 55, highlighting the need for novel therapeutic strategies. Hypoxia-inducible factor 2 alpha (HIF-2α) has been genetically validated as a critical driver of colorectal tumorigenesis, with intestinal epithelium–specific deletion in mice markedly reducing tumor formation. While selective HIF-2α inhibitors such as PT2385 are FDA-approved for renal cell carcinoma, pharmacologic HIF-2α inhibition has not been explored in CRC. Here, we demonstrate that HIF-2α inhibition alone fails to suppress CRC growth in vitro under normoxic or hypoxic conditions and in xenograft models in vivo. To identify vulnerabilities induced by HIF-2α blockade, we performed an unbiased CRISPR metabolic screen, revealing cholesterol biosynthesis as a critical dependency. Targeting this pathway with clinically approved statins (atorvastatin, pitavastatin, simvastatin) synergized with PT2385 to suppress CRC cell growth, reduce colony formation, and enhance cell death. Mechanistic studies show that combined HIF-2α and HMG-CoA reductase inhibition promotes ferroptosis, characterized by increased lipid peroxidation and depletion of antioxidant metabolites. These effects are fully reversed by the ferroptosis inhibitor liproxstatin-1. Genetic knockdown of HIF-2α or HMG-CoA reductase recapitulated the enhanced sensitivity to combination therapy. In vivo, co-administration of PT2385 and atorvastatin significantly reduced tumor growth and increased ferroptotic cell death in xenografts, confirming the mechanistic link. Collectively, these findings uncover a metabolic vulnerability of CRC to dual HIF-2α and cholesterol biosynthesis inhibition, supporting a clinically actionable strategy that leverages safe, FDA-approved statins to potentiate HIF-2α-targeted therapy.

Enhancer elements that reside within 3 ′ untranslated regions (UTRs) are an understudied phenomenon. Given the independent regulatory functions of enhancers and 3 ′ UTRs - enhancers governing pre-transcriptional control of gene expression and 3 ′ UTRs mediating post-transcriptional regulation of messenger RNA (mRNA) fate - 3 ′ UTR-associated enhancers may integrate these complementary layers to coordinate gene expression across multiple regulatory stages. Non-coding variation, impacting regulatory DNA, underpins the genetic architecture of disease. Indeed, the vast majority of single nucleotide polymorphisms (SNPs) associated with human complex diseases map to the non-coding genome, with causal variants particularly enriched within enhancers. Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder associated with non-coding risk variants, many of which are increasingly linked to enhancer disruption. The CAV1 gene, encoding the neuroprotective protein Caveolin-1, is a known ALS risk gene, yet the functional consequences of ALS-associated variation in its regulatory elements remain largely unexplored. Here, we combine genome-wide enhancer profiling with targeted experimental validation to define a previously uncharacterised ALS-associated enhancer embedded within the CAV1 3 ′ UTR, systematically assess its regulatory potential, and evaluate the impact of ALS-associated SNPs on enhancer function. We show that an individual ALS-associated SNP within this 3 ′ UTR-associated enhancer may disrupt function on multiple levels: at the DNA and chromatin level, by altering transcription factor binding with potential effects on recruitment of epigenetic co-regulators; and at the RNA level, by reshaping the structure and stability of a novel enhancer RNA transcribed from this locus. Collectively, our findings highlight this proximal CAV1/CAV2 enhancer as a functionally important regulatory element embedded with the 3 ′ UTR of an ALS risk gene, illustrate how non-coding variants can impact multiple layers of gene regulation, and provide mechanistic insight into how intragenic enhancers contribute to ALS risk. More broadly, this work underscores the importance of 3 ′ UTR-associated enhancers as modulators of risk gene expression and underexplored contributors to human complex disease.

Background Current whole-cell killed oral cholera vaccines have utility but require multiple doses and have limited efficacy in young children. PanChol is a single-dose live-attenuated cholera vaccine derived from the current seventh pandemic Vibrio cholerae O1 strain. It co-expresses Inaba and Ogawa antigens, over-expresses the non-toxic cholera toxin B subunit, and is designed to minimize reactogenicity and prevent toxigenic reversion. We assessed safety and immunogenicity in a first-in-human trial. Methods In a dose-escalation phase at Brigham and Women’s Hospital (Boston, MA, USA), seven cohorts received one dose of 10 4 -10 10 colony-forming-units (CFU) PanChol. Two dosing groups of 2×10 7 and 2×10 8 were subsequently evaluated in a double-blind, placebo-controlled module. Fecal shedding was assessed until day five; safety and immunogenicity were monitored for six months. This trial is registered with ClinicalTrials.gov , NCT05657782 . Findings Between Dec 2022 and Feb 2025, 57 healthy adults were enrolled (dose-escalation: n=21; expansion n=28 vaccine and n=8 placebo recipients). PanChol was safe and well-tolerated at all doses, and no safety concerns were identified including no vaccine-related serious adverse events. In the dose-escalation phase, 81% (17/21) of participants had 39 unsolicited adverse events (AE). In the randomized module, at least one AE occurred in 64% (9/14) at 10 7 dose and in all 10 8 (13/13) and placebo (7/7) recipients. Most AEs were mild and only four were >grade 2 (all unrelated to vaccine). Shedding was detected in 44 recipients of ≥10 5 CFU, with no relationship to dose. All 45 vaccinees given ≥10 5 CFU seroconverted vibriocidal antibodies to both serotypes, with comparable mean titers across doses. IgM responses targeting Inaba or Ogawa polysaccharides were detected in 44 and 41 vaccinees respectively, and anti-toxin IgG responses were measured in 21 vaccinees. Antibody lymphocyte supernatant assays demonstrated mucosal IgA to these antigens and to colonization factor, TcpA. Interpretation A single oral dose of PanChol induced 100% vibriocidal seroconversion over a 100,000-fold dose range with no safety concerns. These findings support further development of PanChol as a new tool for cholera prevention, including studies in endemic settings and in children. Funding Wellcome Trust. Research in context Evidence before this study Cholera remains a global public health threat; killed oral cholera vaccines (OCVs) are important for control. However, they have limited efficacy in young children and require multiple doses for maximum efficacy. Live-attenuated OCVs, like natural infection, may induce protective immunity with a single dose. While other live-attenuated OCVs have been developed, none are WHO prequalified. We searched PubMed from inception to July 2025 for studies evaluating OCVs using the terms “live oral cholera vaccine trial O1”, yielding 25 non-review clinical trial articles. All tested live vaccines were derived from the extinct classical V. cholerae biotype or early El Tor biotype strains and were Inaba serotype. None were engineered to be resistant to reversion to toxigenicity. Added value of this study This first-in-human trial establishes that PanChol, a live-attenuated OCVs engineered from the current global pandemic El Tor V. cholerae O1 strain, is safe and immunogenic in an adult population in Boston, USA. Unlike previous live-attenuated vaccines, PanChol expresses both Inaba and Ogawa serotype antigens, is engineered for enhanced genetic stability, and resists toxigenic reversion. PanChol shedding, a marker for vaccine replication in the intestine, was detectable across doses 10 5 -10 10 CFU. Whole genome sequencing of PanChol isolated from vaccinees’ stool confirmed the vaccine’s genomic stability. Across all doses, 100% of vaccinees seroconverted to both Inaba and Ogawa serotypes, demonstrating potent immunogenicity. Implications of all the available evidence PanChol’s favorable safety profile and immunogenicity support additional development as a new agent for cholera control. Since PanChol is derived from the current pandemic strain, and natural infection stimulates more potent immunity to cholera than killed vaccines, PanChol may offer effective single-dose protection for children. It may be beneficial for reactive vaccination campaigns and for alleviating the global shortage of killed OCVs. These positive results warrant the establishment of the vaccine’s safety and immunogenicity in cholera endemic settings and age de-escalation trials.

Tunnels in deep underground mines provide a unique interface between the surface and deep subsurface habitats. There, microbes from the surface may enter the deep tunnels as surface air is drawn into the deep mine tunnels to provide ventilation. This extreme hosting environment provides a condition for microbes to develop novel capabilities, such as the production of natural products of biotechnology or medicinal importance. This study characterized the genomes of four novel isolates from deep subsurface biofilms of the previously abandoned gold mine, which was used as a model for the underground study. Here the microbiome samples were obtained from thin, whitish, glistening biofilm samples naturally formed on the rock walls (1478 meters deep at SURF). These samples are herein referred to as “cave silver” biofilms. The samples provide 10 GB of high-quality whole genome sequences that were assembled into contigs/scaffolds and structurally and functionally annotated against various databases. Subsequently, the genomes were analyzed for biosynthetic gene clusters (BGCs) encoding secondary metabolites of biotechnology and medical importance using the antiSMASH and the NaPDoS web servers, respectively. In brief, the assemblies produced four drafted genomes of different lengths and annotated features for each strain’s genome, including gene clusters involved in the quorum sensing (QS) pathway. Several BGC-encoded secondary metabolites of natural products or compounds such as polyketides (PKS, PKS III, ketosynthase domain-KS), non-ribosomal peptides (condensation domain, NRPS), and terpenoids (terpenes I, polyenes type II), were identified. Furthermore, many overexpressed enzymes, most of which are shared among the strains and some unique to individual bacteria strains, were identified, revealing possible shared and individualized strain activities in the biofilms during the sample collection. CRISPR peptides were also detected in three of the four bacteria strains. In this study, we examine the genomes of microbes colonizing extreme subsurface environments and how these conditions promote the development of microbial systems that are potentially capable of producing medicinally natural products and secondary metabolites that may be used to enhance advancements in biotechnology products. Abstract Figure Importance Deep subsurface environments host unique microbial communities with specialized adaptations. By characterizing genomes from “cave silver” biofilms, this study reveals biosynthetic gene clusters and pathways for secondary metabolite production, including polyketides, peptides, and terpenoids. These findings highlight the potential of extreme-environment microbes as a novel source of biotechnologically and medically valuable natural products.

Bacteriophages and bacteria engage in a continuous evolutionary arms race, driving the development of intricate bacterial defense systems such as CRISPR-Cas, BREX, Gabija, and Shedu. Here, we characterize a two-component KELShedu system in Escherichia coli that confers resistance to phages via abortive infection. The KELShedu system comprises KELA, a dsDNA-binding protein, and KELB, a metal ion-dependent nuclease harboring the DUF4263 domain. In addition, we find that physiological levels of NTP inhibit the DNA cleavage activity of the KELShedu system, suggesting that KELShedus activation depends on reduced intracellular NTP levels during phage invasion. Our research demonstrates that the KELShedu system responds to nucleotide depletion triggered by phage replication, leading to non-specific degradation of cellular DNA and ultimately inducing abortive infection. These insights into the KELShedu system expand the repertoire of bacterial anti-phage mechanisms and lay the groundwork for novel applications in microbial engineering and therapeutic development.

Summary The use of alternative promoters and splicing increases molecular complexity and diversifies cellular functions. However, mechanisms of crosstalk between transcription and splicing remain poorly understood. Here, we utilize CRISPR epi-editing in neurons to manipulate isoforms of the synaptic organizer, Neurexin-1, and elucidate mechanisms underlying the co-regulation of alternative promoters and splicing. Surprisingly, silencing individual Neurexin-1 promoters altered downstream promoter activity via transcriptional interference and biased splicing decisions. Our data reveals transcriptional interference as key to shaping cell type-specific Neurexin-1 isoforms in the mouse hippocampus and demonstrates the power of epi-editing to uncover regulatory interactions between RNA processes in the brain.

CRISPR-Cas13d is increasingly used for RNA knockdowns due to its programmability, but off-target RNA binding and cleavage of near-cognate RNAs hinder its broader adoption. Here, we explore the mechanisms of nuclease activation by solving seven ternary cryo-electron mi-croscopy structures of wild-type Cas13d in complex with matched and mismatched targets. These structures reveal a series of active, intermediate, and inactive states that illustrate a detailed activation mechanism. The crRNA undergoes dramatic conformational changes upon target RNA binding, with the helical-1 domain transitioning from an initially docked state with the N-terminal domain to an allosterically switched conformation that stabilizes the RNA duplex. Quantitative kinetics reveal that a single proximal mismatch preserves nanomolar binding affinity but completely abolishes nuclease activity by trapping Cas13d in an inactive state. We identify an active site loop in the HEPN domains that regulates substrate accessibility, with alanine scanning mutagenesis revealing both hypo- and hyperactivated variants. These findings establish the structural basis for Cas13d’s exquisite mismatch surveillance and provide a mechanistic framework for engineering RNA-targeting specificity and activity across HEPN nuclease family members.

The balance between excitatory and inhibitory neurotransmission is fundamental for normal brain function, yet the adaptation of individual neurons to disrupted excitatory-inhibitory balance is not well understood. We developed highly efficient, in vivo RNA electroporation-based single-cell gene editing to investigate neuronal responses to loss of fast inhibition. Using CRISPR-Cas9 components delivered as RNA, we knocked out GABA-A receptor β subunits in individual layer 2/3 cortical neurons in mouse visual cortex, eliminating fast inhibition. In vivo patch-clamp recordings revealed that cortical neurons adapted to inhibition loss through two sequential mechanisms: a transient reduction of excitatory synaptic input, followed by intrinsic membrane property changes that decreased input resistance. This sequential adaptation program ultimately prevented target neurons from contributing spikes to the cortical network. Our RNA-based single-cell gene editing approach enables investigation of cellular responses independent of network effects, providing new insights into neuronal homeostasis and gene function in individual cells in vivo.

Abstract Background: The plant circadian clock is crucial for regulating developmental and metabolic processes, enabling crops to adapt to environmental changes and maintain high productivity. In rice, the clock gene OsPRR37 plays a pivotal role in photoperiod sensitivity and the regulation of yield-related traits. However, the complete regulatory network of OsPRR37 remains largely unexplored. Results: This study utilized an integrated multi-omics approach, combining transcriptome profiling, DNA affinity purification sequencing (DAP-seq), and protein–protein interaction (PPI) mapping to construct a multi-layered regulatory model of OsPRR37 . CRISPR/Cas9 knockout lines showed significant changes in flowering time, plant height, panicle architecture, and spikelet number. Transcriptome analysis associated OsPRR37 with pathways related to photosynthesis, carbohydrate metabolism, and stress responses. Comparative analysis of knockout and overexpression datasets identified 454 candidate target genes exhibiting inverse expression patterns, including regulators of flowering and chlorophyll biosynthesis. DAP-seq revealed 1,679 high-confidence DNA-binding sites, with nine genes identified as direct targets, six of which contained conserved motifs associated with cytokinin signaling, inflorescence architecture, and meristem determinacy. PPI mapping through a yeast two-hybrid screen identified 26 interacting proteins, including OsGlyRS3 and OsSnRK1A, which are involved in flowering, sugar signaling, chloroplast development, and hormone metabolism. Structural modeling suggested that OsGlyRS3 may stabilize OsPRR37 protein complexes, while OsSnRK1A could modulate its DNA-binding capacity under sugar-deficient conditions. Conclusions: The findings establish OsPRR37 as a central regulatory hub that coordinates flowering, energy metabolism, chloroplast function, and stress adaptation through a hierarchical network comprising a Modulatory Layer of protein interactors, a Direct Target Layer of DNA-bound genes, an Indirect Coherent Layer of transcriptional cascades, and a Diffuse Response Layer encompassing broad metabolic outputs. This model provides a comprehensive framework for understanding how OsPRR37 integrates circadian signals to control multiple agronomic traits and offers valuable targets for breeding climate-resilient, high-yielding rice varieties.

Abstract Triple-negative breast cancer (TNBC) is a subtype with limited treatment options and poor outcomes, particularly in the metastatic setting. Although immunotherapy has shown efficacy in early-stage disease, its benefit remains suboptimal in women with locally advanced and metastatic TNBC. Here, we identify the splicing factor PTBP1 as a tumor-intrinsic regulator of immune evasion in metastatic TNBC. By integrating clinical, single-cell, and bulk transcriptomic data with multiplex immunohistochemistry, CRISPR-Cas9 genome editing, and functional assays, we show that PTBP1 impairs antigen presentation, promotes T cell dysfunction, and is associated with worse outcomes, independent of tumor-infiltrating lymphocyte levels. Furthermore, CRISPR-mediated silencing of PTBP1 restores HLA expression and reactivates antigen presentation pathways in TNBC. PTBP1 expression is elevated in metastatic compared to primary TNBC tumors and correlates with immune dysfunction signatures. Consistently, in the phase II TONIC clinical trial, metastatic TNBC patients with PTBP1-high tumors had poor response and shorter survival following PD-1 blockade, and PTBP1 expression showed a predictive performance comparable to PD-L1 and TILs in this cohort. These findings position PTBP1 as a tumor-intrinsic regulator of immune evasion and a potential biomarker to inform immunotherapy strategies in metastatic TNBC.

Abstract Background RFX3 is a transcription factor (TF) critical for pancreatic endocrine development. Its loss impairs β-cell differentiation, promotes enterochromaffin cell (EC) generation, and induces apoptosis. However, the role of non-coding RNAs in mediating these effects remains poorly understood. Methods Using CRISPR/Cas9-derived RFX3 knockout (KO) human iPSC-derived pancreatic islets, we performed integrated transcriptomic analyses of coding and non-coding RNAs. Differentially expressed miRNAs (DEmiRs) and lncRNAs (DElncRNAs) were validated by RT-qPCR. Target prediction and competing endogenous RNA (ceRNA) network analyses were conducted to explore potential regulatory interactions affecting key endocrine genes. Results RFX3 deficiency induced widespread transcriptomic changes in human iPSC-derived pancreatic islets. Core β-cell markers and key pancreatic TFs were downregulated, alongside genes regulating ion channels, vesicle trafficking, metabolic sensing, and insulin secretion. Conversely, apoptotic and EC genes were upregulated. RFX3 KO islets exhibited significant alterations in miRNA profiles, including upregulation of miR-451a, miR-215-5p, miR-122-5p, miR-338-3p, miR-194-5p, miR-378a-3p , and the miR-29 family, which are predicted to target critical pancreatic endocrine genes. Several lncRNAs, including LINC00461, MIAT, RMST , and AC020916.1 , were downregulated, potentially influencing miRNA activity via ceRNA interactions. Integrated analyses identified core regulatory axes, such as miR-4455/INS, miR-122-5p/ARX, and miR-660-3p/GHRL, while apoptosis-related genes, including CASP, TNFSF10 , and TXNIP , were also predicted targets of dysregulated miRNAs. Conclusions Our findings reveal widespread alterations in non-coding RNA networks associated with RFX3 loss, highlighting a potential layer of post-transcriptional regulation linked to impaired pancreatic endocrine development. These results provide insights into how RFX3 deficiency may reshape islet transcriptomes, influencing β-cell maturation, function, and survival.

Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the Leptospira genus. Genome editing in Leptospira has been difficult to perform. Currently, the functionality of the CRISPR-Cas system has been demonstrated in species such as Leptospira interrogans. However, the different CRISPR-Cas systems present in most of the 77 species are unknown. Therefore, the objective of this study was to identify the CRISPR-Cas systems present in the genomes of the Leptospira genus using bioinformatics tools. Methods: bioinformatics workflow was followed: the genomes were downloaded from the NCBI database, Cas proteins detection was carried out using the CRISPR-CasFinder and RAST web servers, functional analysis of Cas proteins (InterProScan, ProtParam, Swiss Model, Alphafold3, Swiss PDB Viewer, and Pymol), conservation pattern detection (MEGA12, and Seqlogos), spacer identification (Actinobacteriophages db and BLAST), and bacteriophage detection (Phaster, and Phastest). Results: Cas proteins were detected in 36/77 species of the Leptospira genus, these proteins were (Cas1-Cas9, and Cas12). The proteins were classified into class 1 and class 2 systems, and types I, II, and V. Direct repeats and spacers were detected in 19 species. The direct repeats presented two nucleotide conservation motifs. With the spacer sequences, 270 different bacteriophages were identified. Three intact bacteriophages were detected in the genome of four Leptospira species. Two saprophytic species have complete CRISPR-Cas systems. Conclusions: The presence of Cas proteins, direct repeats, and spacer sequences homologous to bacteriophage genomes suggests a functional CRISPR-Cas system in at least 19 species.

Mating in insects commonly induces an alteration in behavior and physiology in the female that ensures optimal offspring. This is referred to as a post-mating response (PMR). The induction of a PMR requires not only male-derived factors transferred with semen during copulation, such as sex peptide (SP) in Drosophila , but also intrinsic female signaling components. The latter signaling remains poorly understood in most insects, including the brown planthopper (BPH) Nilaparvata lugens , a devastating rice pest. In BPHs the PMR comprises a reduced receptivity to re-mating and increased oviposition. Here, we demonstrate that the neuropeptide corazonin (CRZ) and its receptor (CrzR) are critical for the PMR in female BPHs. Peptide injection and knockdown of CRZ expression by RNAi or CRISPR/Cas9-mediated mutagenesis demonstrate that distensible CRZ signaling suppresses mating receptivity in virgin N. lugens females and mediates a reduction in re-mating frequency and increased ovulation. The CrzR is highly expressed in the female reproductive tract, and CrzR -knockdown phenocopies Crz diminishment. Importantly, female CRZ/CrzR signaling is indispensable for male seminal fluid factors (e.g. maccessin) to induce the PMR. With disrupted CrzR signaling, seminal fluid or maccessin injection fails to reduce female receptivity. Notably, CRZ is not produced in male accessory gland (MAG) and thus not transferred during copulation. However, male Crz knockout impairs the PMR in mated females and combining male and female Crz knockouts nearly abolished the PMR. Transcriptomics of the MAG indicates that Crz knockout affects the expression of numerous seminal fluid protein genes. Finally, we found that also in female Drosophila melanogaster , disrupted Crz signaling resulted in increased re-mating and reduced oviposition, while CRZ injection suppressed virgin receptivity and increased oviposition. In summary, our study reveals that endogenous female CRZ signaling and male-derived signals cooperate to regulate post-mating transitions in BPHs and Drosophila .

Wheat ( Triticum aestivum L.) is one of the most important crops worldwide, supplying a major share of calories and protein for the global population. Incorporating gene editing into breeding programs is critical to improve yield and stress tolerance, yet wheat remains difficult to transform and regenerate efficiently. These bottlenecks limit the full application of CRISPR/Cas9 for improvement yield in wheat. To address this, transformation parameters were optimized for three methods: immature embryo transformation, callus transformation, and injection-based in planta transformation. Systematic optimization of Agrobacterium strain, bacterial density, acetosyringone concentration, and incubation conditions resulted in substantially improved transformation success. Efficiencies of 66.84% for immature embryos, 55.44% for callus, and 33.33% for in planta transformation were achieved, representing more than tenfold increase compared with previously reported rate of ∼3%. A key innovation was the shortening of the callus induction stage for immature embryos, reducing the time required for plant regeneration by approximately one month while maintaining high transformation efficiency. The protocols were validated through CRISPR/Cas9-mediated knockout of TaARE1-D , a negative regulator of nitrogen uptake and yield. Generated mutants exhibited increased grain number, spike length, grain length, and thousand-grain weight, as well as the characteristic stay-green phenotype associated with loss of TaARE1-D function. The optimized protocols provide robust platforms to accelerate gene-editing in wheat to increase yield and stress-tolerance.