Cytotoxic CD8 + T-cells play central roles in tumor immunotherapy. Understanding mechanisms that regulate development, differentiation, and functions of cytotoxic CD8 + T-cells leads to development of better immunotherapies. By combining primary T-cell culture and a syngeneic mouse tumor model with both genome-wide and custom CRISPR/Cas9 screenings, we systematically identified genes and pathways that regulate PD-1 expression and functions of CD8 + T-cells. Among them, inactivation of a key enzyme in glycoconjugate biosynthesis, beta 1, 4-galatosyltransferase 1 (B4GALT1), leads to significantly enhanced T-cell receptor (TCR) activation and functions of CD8 + T-cell. Interestingly, suppression of B4GALT1 enhances functions of TCR-T-cells, but has no effect on chimeric antigen receptor T (CAR-T) cells. We systematically identified the substrates of B4GALT1 on CD8 + T-cell surface by affinity purification and mass spectrometry analysis, which include protein components in both TCR and its co-receptor complexes. The galactosylation of TCR and CD8 leads to reduced interaction between TCR and CD8 that is essential for TCR activation. Artificially tethering TCR and CD8 by a TCR-CD8 fusion protein could bypass the regulation of B4GALT1 in CD8 + T-cells. Finally, the expression levels of B4GALT1 normalized to tumor infiltrated CD8 + T-cells in tumor microenvironment are significant and negatively associated with prognosis of human patients. Our results reveal the important roles of protein N-glycosylation in regulating functions of CD8 + T-cells and prove that B4GALT1 is a potential target for tumor immunotherapy.

Although glucocorticoids are widely used to alleviate side effects of prostate cancer (PCa) treatment, the glucocorticoid receptor (GR) exhibits a dual role exerting tumor-suppressive effects by inhibiting early-stage PCa cell proliferation, while also promoting oncogenic progression by mediating antiandrogen resistance. The mechanisms underlying this functional dichotomy have remained elusive and poorly characterized. Using genome-wide analyses and CRISPR-based genome editing, we identified the tumor protein p63 as a key mediator of GR’s tumor-suppressive chromatin activity. Loss of p63 reprograms GR activity toward an oncogenic state, marked by enhanced cell migration, invasion, and altered morphology. This shift is driven by increased GATA2 expression, which alters GR’s chromatin binding and transcriptional output. Together, our findings uncover a p63–GATA2 molecular switch that governs the dual role of GR in PCa, establishing transcription factor crosstalk as a critical regulator of GR-driven oncogenic reprogramming and cellular plasticity.

Chimeric antigen receptor (CAR) T cell therapies are being widely investigated in both autologous and allogeneic settings, with gene editing providing new strategies to address barriers to mismatched cell therapies. Currently ‘universal’ donor derived T cell therapies require intensive lymphodepletion and are prone to host-mediated rejection. CD38, a transmembrane glycoprotein involved in cell activation and bioenergetics, is a promising immunotherapy target for haematological malignancies. Disruption of CD38 expression using base editing prevented fratricide between T cells expressing anti-CD38 CAR (CAR38). Additional base editing enabled generation of a ‘universal’ donor CAR38-T cells, devoid of endogenous TCRαβ and Human Leukocyte Antigen (HLA) molecules after disruption of T Cell Receptor Beta Constant ( TRBC ), Beta-2 microglobulin ( B2M ), and Regulatory Factor X5 ( RFX5 ). Removal of cell surface HLA expression enabled evasion of anti-HLA antibodies in sera from sensitised donors and reduced allo-stimulation in mixed lymphocyte cultures (MLCs), while TCRαβ disruption prevented allo-reactivity. In MLCs, CAR38 expression enabled potent ‘allo-defense’ activity against CD38 + allo-reactive cells. Multiplex-base-edited CAR38-T cells exhibited antigen-specific anti-leukemic activity against human B, T, and myeloid malignancies and inhibited disease progression in humanised murine xenograft models. CAR38-T cells offer a potent ‘off-the-shelf’ strategy against CD38 + haematological malignancies and plasma cells associated with autoimmunity.

SUMMARY Inherited mutations in VPS35 and the kinase LRRK2 lead to hyperphosphorylation of Rab GTPases and promote the formation of phospho-Rab signalling complexes. A subset of RH2 domain-containing proteins from the RILP-homology family, including RILP, RILPL1, RILPL2, JIP3, and JIP4 are Rab effectors that recognize the LRRK2-phosphorylated switch 2 threonine of phospho-Rab8A and phospho-Rab10. More recently, phospho-Rabs have been found on lysosomal membranes within multi-protein assemblies involving TMEM55B and RILPL1. TMEM55B is a 284-residue lysosomal membrane protein with no homology to known proteins. It comprises a 218-residue cytosolic N-terminal region and two predicted transmembrane α-helices. Residues 80– 160, which face the cytosol, mediate binding to a C-terminal motif of RILPL1, formed after RILPL1 associates with phospho-Rab8A. Here, we report the crystal structures of TMEM55B alone and in complex with a C-terminal RILPL1 peptide, encompassing the TMEM55B interaction region, which we define as the TMEM55B Binding Motif (TBM). The cytosolic domain of TMEM55B adopts a rigid architecture of two tandem RING-like domains, each forming a Zn²⁺-stabilized 40-residue β-sandwich. TBM binding is mediated primarily by backbone hydrogen bonding and anchored by two glutamate residues from RILPL1. These findings support a model in which RILPL1 is recruited to phospho-Rab8A–positive lysosomes prior to TMEM55B engagement. Further co-immunoprecipitation and mutational analyses indicate that TMEM55B forms complexes independently of phospho-Rabs with proteins containing a conserved TBM, like that of RILPL1, including JIP3, JIP4, OCRL, WDR81, and TBC1D9B. Together, these findings uncover previously unrecognized regulatory networks associated with TMEM55B and lysosomal function and suggest that TMEM55B serves as a central hub for adaptor recruitment at the lysosomal membrane.

Friedreich ataxia (FRDA) is a progressive neuromuscular degenerative disorder caused by GAA repeat expansions in the FXN gene, leading to frataxin deficiency and multisystem pathology. Cardiomyopathy is the leading cause of mortality in individuals with FRDA. To investigate the cellular and molecular mechanisms underlying FRDA-associated cardiac dysfunction, we employed induced pluripotent stem cell (iPSC) lines derived from three individuals with FRDA, each paired with an isogenic control line generated through CRISPR/Cas9-mediated excision of the pathogenic GAA repeat expansion. Correction of the mutation restored FXN expression to levels comparable to healthy donor iPSCs, and all lines differentiated efficiently into cardiomyocytes. Functional analysis revealed significant contractile abnormalities in FRDA cardiomyocytes and multicellular cardiac microtissues, including prolonged contraction and relaxation times and faster beating rates, consistent with clinical observations of cardiac contractile dysfunction. FRDA cardiomyocytes also exhibited pathological features such as increased cell size, irregular calcium transients, elevated mitochondrial reactive oxygen species levels, increased mitochondrial fission and increased cell death. These phenotypes were exacerbated by pathological levels of iron supplementation in culture media, highlighting the heightened sensitivity of frataxin-deficient cardiomyocytes to iron-induced metabolic stress. RNA sequencing revealed a distinct transcriptional profile associated with frataxin deficiency. MEG3 and PCDHGA10 were consistently dysregulated across all three FRDA-iPSC lines and may represent early molecular markers of FRDA cardiomyopathy. Together, these findings establish a robust human iPSC model of FRDA cardiomyopathy that captures early disease phenotypes and reveals novel molecular targets. This preclinical human model provides valuable insight into the pathogenesis of FRDA and provides a platform for developing early-stage therapeutic interventions.

The alternative splicing of the adapter protein NUMB is dysregulated in multiple cancer types, regulating its functional divergence towards either tumor suppression or oncogenesis in an isoform dependant manner. Here we utilized a NUMB exon 9 (E9) splicing reporter in a genome-wide CRISPR screen to identify splicing regulators SRRM1 and SRSF11 that promote NUMB oncogenic splicing in colorectal, lung and breast cancer cell lines. Furthermore, SRRM1 and SRSF11 share common protein interactors, RNA targets and effects on an oncogenic splicing program which favors the expression of pro-tumorigenic isoforms. In addition to NUMB E9, SRRM1 regulates oncogenic splicing events in genes encoding signaling proteins, transcription factors and actin cytoskeleton regulators, many of which also undergo developmentally regulated splicing, including CD44, MKNK2, ECT2, DIAPH1, KAT5, TCF7L2, FOXM1 and TBX3,. Loss of SRRM1 in colon cancer cells reduces cell proliferation and colony formation capabilities as well as expression of tumour promoters Cyclin D1, Notum, and PRDX2. Our data indicate that SRRM1 regulation of alternative splicing represents a node to target multiple properties of malignant cells, with broad effects on cellular signaling, proliferation, EMT, apoptosis resistance and stemness.

Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells offer a promising platform for off-the-shelf immunotherapy against hematological malignancies. NK cell function is dynamically regulated through education driven by inhibitory receptors, including CD94/NKG2A and killer cell immunoglobulin-like receptors (KIR). However, the acquisition of inhibitory receptors in iNK cells and their role during differentiation and education remains poorly defined. In this study, we monitored receptor repertoires, transcriptional states, and functional responses in a range of genetically engineered iNK cell lines. Transcriptional reference mapping placed iNK cells close to cytokine-activated NKG2A+ CD56dim peripheral blood (PB) NK cells. Despite their early differentiation stage, iNK cells displayed a well-developed cytotoxic effector program, which was also reflected in high protein expression of Eomes, granzyme B, and activating receptors DNAM-1 and NKG2D. Acquisition of NKG2A by iNK cells was associated with a more differentiated transcriptional state and superior functional responses against a broad range of targets, including those expressing low to moderate levels of HLA-E, suggesting attenuated inhibitory signaling through NKG2A in iNKs. CRISPR knockout of B2-microglobulin (B2M) in iNK cells revealed that the functional potency of NKG2A+ iNK cells was independent of educating interactions with HLA-E in cis or trans. Finally, CRISPR-mediated ablation of NKG2A led to a spontaneous compensatory surface expression of CD94/NKG2C heterodimers, associated with enhanced IFN-g production and cytotoxic activity against target cells with forced high expression of single-chain B2m-HLA-E-peptide trimers. Our results indicate an education-independent functional maturation of iNK cells, characterized by potent effector programs coupled with a favorable early-stage transcriptional profile.

ABSTRACT Pumilio (PUM) RNA binding proteins are crucial for regulating gene expression by binding to a conserved motif in the 3′-untranslated region (3′-UTR). Despite their importance, the role of PUM in plants is largely unexplored. Here, we investigated the functions of Arabidopsis group I PUMs (APUM1-APUM6), which are ubiquitously expressed and localized in the cytosol. While single apum mutants exhibit no visible phenotypes, CRISPR/Cas9-generated apum1 apum2 apum3 triple mutants ( apum1/2/3 ) display reduced growth in both vegetative and reproductive organs, alongside hypersensitivity to various stresses. Remarkably, apum1/2/3/4 quadruple mutants are embryonically lethal, highlighting their essential role in embryo development. Transcriptomic profiling revealed that differentially expressed genes (DEGs) upregulated in apum1/2/3 are enriched in pathways related to photosynthesis, stress responses and anthocyanin biosynthesis, while downregulated DEGs are associated with biotic stress defense and hydrogen peroxide metabolism. This suggests that APUM1/2/3 act as molecular hubs balancing plant growth and stress adaptation. Biochemical assays showed that recombinant APUM homologous domains bind to the 5’-UG U GUAUA-3’ core motif in the 3’-UTR of the transcription factor Production of Anthocyanin Pigment1 (PAP1), crucial for anthocyanin biosynthesis. Notably, single nucleotide substitutions, except for the third U, do not affect binding, while multiple mutations disrupt interaction. Consistently, apum1/2/3 mutants accumulate significantly more anthocyanin than wild-type plants. Furthermore, we predicted 7053 potential target genes for APUM1/2/3, with 1609 present among the upregulated DEGs in apum1/2/3 . Taken together, our findings demonstrate that group I APUMs are vital posttranscriptional regulators, providing a new perspective on the trade-offs between growth and stress resilience in plants.

Internal ribosome entry sites (IRESs) enable cap-independent initiation of picornaviral RNA translation and, together with canonical translation initiation factors, typically require specific cellular proteins known as IRES trans -acting factors (ITAFs). While the type II IRES of foot-and-mouth disease virus (FMDV, an aphthovirus) has been shown to depend on the oncoprotein ITAF45, also known as Proliferation-Associated 2G4 (PA2G4) or ErbB-3 receptor Binding Protein (EBP1), for in vitro assembly of the 48S pre-initiation complex, some related type II IRESs, such as that of encephalomyocarditis virus (EMCV, a cardiovirus), can form the initiation complex independently of ITAF45. In this study, we performed a genome-wide CRISPR screen and identified knockouts of PA2G4 / EBP1 / ITAF45 in cells that survive EMCV infection, suggesting an important role for this factor. We show that the p48 isoform of ITAF45, but not the p42 isoform, is crucial for efficient EMCV/Mengovirus replication and for propagation of replicons in human cell culture. Loss of ITAF45 markedly diminishes EMCV and FMDV IRES activities, which can be rescued by re-expression of ITAF45-p48. Interestingly, cell-free translation assays reveal that EMCV IRES activity is less ITAF45-dependent in vitro , in contrast to FMDV, raising questions about the versatile functions of ITAFs in IRES-driven translation. These findings reveal an isoform-specific function of ITAF45 in supporting cardiovirus infection and provide new insights into the complex regulation of IRES-driven translation, with implications for developing targeted antiviral strategies.

Mutations in the SOD1 gene are among the most significant genetic contributors to amyotrophic lateral sclerosis (ALS), with different variants linked to varying disease severity. To investigate the molecular mechanisms driving this variability, we conducted RNA sequencing on spinal motor neurons (MNs) differentiated from isogenic human induced pluripotent stem cell (iPSC) lines engineered via CRISPR/Cas9. These lines carried two representative SOD1 heterogenous mutations, D91A and G94A, and were analyzed at Days 10 and 20 of neuronal maturation stage to capture the temporal changes of gene expression. We aim to explore how these mutations affect MN function, identify distinct molecular pathways that may explain the variable severity of ALS, and investigate the role of translation and metabolic dysregulation in disease progression.

Genes that enhance T cell function represent promising targets for improving engineered T cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T cell persistence, employing Sleeping Beauty ( SB ) insertional mutagenesis, which induces both gain-(GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked. We developed transgenic mice carrying D oxycycline (Dox)-inducible SB mutag e nesis s y stem (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T cell persistence under chronic antigen exposure. Specifically, CD8⁺ T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8⁺ T cells using enhanced-specificity tagmentation sequencing (esTag-seq) and RNA-seq, respectively. Under chronic stimulation, SB -mutagenized CD8⁺ T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation stress. Among these, T2/Onc2 insertions into Bach2 and Elmo1 were repeatedly found at the genomic level and were associated with altered nascent transcript expression. Bach2 , known as a key regulator of T cell memory formation and resistance to chronic viral infection but less characterized in engineered T cells for cancer therapy, was found to enhance in vivo tumor persistence in the B16-Ova tumor model. We showed that ectopic Bach2 expression levels influence engineered T cell differentiation lineage. A Bach2 low signature allowed differentiation into both KLRG1⁺ and CD62L⁺ phenotypes, whereas Bach2 high restricted differentiation predominantly to the CD62L⁺ subset. Finally, in human CART19-28ζ cells, BACH2 overexpression enhanced cytotoxicity and improved tumor control following chronic cancer stimulation. Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T cell therapeutic phenotypes. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.

Abstract Isoleucine, an essential branched-chain amino acid with broad applications in food, pharmaceuticals, and feed, is predominantly produced via the microbial threonine pathway, which suffers from catalytic complexity and regulatory inefficiency. The propionate pathway offers a streamlined alternative but remains unexplored for L-isoleucine biosynthesis. Here, we engineered Escherichia coli to establish the first propionate pathway-based L-isoleucine cell factory. Critical enzymes—propionyl-CoA synthase (PCS), propionyl-CoA transferase (PCT), and α-ketobutyrate synthase (OBS)—were identified for converting propionate to α-ketobutyrate. Key genes (prpE from Salmonella, pctcp from Gibberella, pctcn from Clostridium propionicum, and nifJ from Moorella thermoacetica) were integrated with the propionate transporter (prpp) and carbonic anhydrase (can) to enhance substrate utilization. ILE-5a, derived from E. coli BW25113 with deletions in brnQ, livJ, and livK, and containing specific insertions, was further modified to create ILE-5b with an additional deletion in yjip. Plasmid-based expression in these strains, ILE-5a and ILE-5b, yielded top producers ILE-5a-P10 and ILE-5b-P10, which achieved L-isoleucine titers of 304 mg/L and 235 mg/L, respectively, in shake-flask fermentation using glucose and propionate as carbon sources. To stabilize production, the optimal gene set (pctcn, nifJ, prpp, can) was genomically integrated via a transposon-encoded CRISPR-Cas system, generating mutants ILE-5a-P11 and ILE-5b-P11. Response surface methodology-optimized medium and 3-L fed-batch fermentation further elevated titers to 1.13 g/L (ILE-5a-P11) and 11.33 g/L (ILE-5b-P11). This study pioneers the propionate pathway for efficient L-isoleucine production, demonstrating its industrial potential through systematic metabolic engineering and process optimization.

Life cycle details or ecological impact are well characterized only for a few ssDNA phages. The Finnlakeviridae family includes one species, Finnlakevirus FLiP . Here, using the same Flavobacterium host and sampling location, we isolated a new strain designated FLiP-2, with 96,7 % genetic identity to the original isolate FLiP. To understand the ecology of this Flavobacterium- infecting phage species, we explored the host interactions of the two FLiP strains and a dsDNA Flavobacterium phage MaF61 under various conditions representing those encountered in their natural habitats in boreal lakes including different temperatures, anoxic conditions, and in the presence of different nutrients and antibiotics. While FLiP and FLiP-2 had similar virion stability outside the host, they exhibited significant differences in plaque morphology and infectivity. FLiP-2 could not replicate in the presence of ampicillin, whereas FLiP thrived even at high concentrations. Both strains of Finnlakevirus FLiP propagated better under or after stress exposure compared to MaF61. Additionally, Finnlakevirus FLiP plaques appeared far from the original infection site, particularly in response to stress, suggesting latent presence within a motile or filamentous bacterium. In conclusion, Finnlakevirus FLiP showed remarkable flexibility in host-interactions being well adapted to fluctuating conditions in boreal freshwaters.

ABSTRACT Acute myeloid leukemia (AML) remains a major therapeutic challenge due to extensive disease heterogeneity and lack of cancer-specific antigens. ADGRE2 has emerged as a promising AML target with broad expression in AML patient blast and leukemic stem cell-enriched populations. However, comparable expression in healthy hematopoietic stem and progenitor cells (HSPCs) and myeloid lineages suggests a high susceptibility to on-target, off-tumor myelotoxicity with ADGRE2-targeted therapies. Guided by human genetics data identifying loss-of-function variants, we evaluated whether ADGRE2 is dispensable in hematopoietic stem cells as a protective approach for transplant-based shielding from ADGRE2-directed therapies. Using CRISPR-Cas9 and adenine base editors, we achieved high-efficiency ADGRE2 knockout (>94%) in HSPCs with corresponding protein loss without impairing cell viability, differentiation, and cytokine release in vitro , or long-term engraftment, multilineage differentiation, and persistence of gene editing in mouse xenografts. We also developed novel ADGRE2-specific chimeric antigen receptor (CAR) T cells that demonstrated potent cytotoxicity against AML cells, even at low antigen levels. Together, these findings establish ADGRE2 as a compelling AML target and provide a framework for hematopoietic stem cell transplant with protective gene editing to enable ADGRE2-directed immunotherapies while minimizing myelotoxicity.

ABSTRACT Heterotrimeric G proteins transduce signals from G protein coupled receptors, which mediate key aspects of neuronal development and function. Mutations in the GNAI1 gene, which encodes Gαi1, cause a disorder characterized by developmental delay, intellectual disability, hypotonia, and epilepsy. However, the mechanistic basis for this disorder remains unknown. Here, we show that GNAI1 is required for ciliogenesis in human cells and use C. elegans as a whole-organism model to determine the functional impact of seven GNAI1 -disorder patient variants. Using CRISPR-Cas9 editing in combination with robust cellular (cilia morphology) and behavioral (chemotaxis) assays, we find that T48I , K272R , A328P , and V334E orthologous variants impact both cilia assembly and function in AWC neurons, M88V and I321T have no impact on either phenotype, and D175V exerts neuron-specific effects on cilia-dependent sensory behaviors. Finally, we validate in human ciliated cell lines that D173V , K270R , and A326P GNAI1 variants disrupt ciliary localization of the encoded human Gαi1 proteins similarly to their corresponding orthologous substitutions in the C. elegans ODR-3 (D175V , K272R , and A328P ). Overall, our findings determine the in vivo effects of orthologous GNAI1 variants and contribute to mechanistic understanding of GNAI1 disorder pathogenesis as well as neuron-specific roles of ODR-3 in sensory biology. ARTICLE SUMMARY Gα subunits of heterotrimeric G proteins transduce signaling from G protein coupled receptors and play important roles in cell communication and complex behaviors. Mutations in the GNAI1 gene, which encodes Gαi1 protein, have been recently linked to a neurodevelopmental disorder; however, it remains unknown how GNAI1 patient mutations disrupt neuronal development or function to manifest in disease. We demonstrate that GNAI1 is required for ciliogenesis and use C. elegans as a whole-animal model in combination with human cells to identify cell-specific and conserved mechanisms of Gα dysfunction.

In C. elegans , the epidermis and its overlying extracellular matrix form a primary protective barrier, functioning as the first line of defense against environmental factors. To properly develop those cellular boundaries, a tightly controlled interaction of many molecules and pathways is needed. Mutant alleles of paqr-2 and iglr-2 (lipid homeostasis), dpy-21 (membrane trafficking), and sma-1 (actin-binding spectrin) result in hermaphrodite tail tip defects suggesting that this simple four-cell structure can serve as a sensitive model for the identification of pathways responsible for the establishment of cellular boundaries. With this in mind, we performed a small forward genetics screen of ∼800 ethyl methanesulfonate-mutagenized haploid genomes and identified 21 mutants with a tail end defective (Ted) phenotype. Whole genome sequencing of these mutants identified mutations in genes encoding either structural constituents of the cuticle itself (mostly collagen genes) or protein with regulatory functions. By using CRISPR/Cas9 we confirmed six novel alleles of ptr-18, paqr-2, nab-1, ncam-1, vab-9 and efn-4. We further characterized the loss of function allele ptr-18(et70) , which encodes a patch domain-containing (PTCHD) protein homologous to human PTCHD1. ptr-18(et70) has a significant effect on growth and development of the worms, while also increasing membrane permeability. Lipidomics analysis revealed no major alterations in membrane lipid composition, implicating cuticle defects as the primary cause of the observed permeability phenotype. Article summary We performed a forward genetics screen to identify hermaphrodite C. elegans mutants with a tail end defect with the goal to discover membrane and morphogenesis regulators. The screen of 800 haploid genomes revealed 21 tail end defective mutants, including 8 novel alleles of interesting regulator protein. We conclude that the tail tip phenotype can be useful in discovery of new pathways and interactions during development.

Uveal melanoma, the most common eye cancer in adults, remains limited to surgical intervention and chemotherapy, with a dismal survival rate that has not improved in over 50 years. To address this therapeutic impasse, we systematically analyzed public gene expression, RNAi, and CRISPR knockout datasets and identified RASGRP3 as an essential gene specifically for uveal melanoma. RasGRP3 is uniquely overexpressed and essential for survival in uveal melanoma cells, but dispensable in healthy cells. RasGRP3 remains “undruggable” due to its intracellular localization and lack of targetable binding pockets. To overcome this, we developed a CRISPR-Cas13d RNA-targeting therapeutic that specifically knocks down RasGRP3 mRNA. This Cas13d-based therapeutic mediates selective uveal melanoma killing through two synergistic mechanisms: (i) direct silencing of the essential RasGRP3 transcript, and (ii) collateral RNA degradation triggered by the cleavage of overexpressed RasGRP3. When delivered via optimized lipid nanoparticles encoding Cas13d mRNA and guide RNA, this strategy eliminated >97% of uveal melanoma cells while sparing healthy cells, including retinal pigment epithelial cells. This approach outperformed conventional Cas9 and siRNA methods in potency without inducing permanent genomic alterations. Our findings establish a RNA-targeting therapeutic for uveal melanoma and a framework for Cas13d-based interventions against broad “undruggable” cancers.

Abstract Background Chimeric antigen receptor (CAR)-based immunotherapies face significant translational challenges in solid tumor applications, particularly regarding manufacturing scalability, tumor targeting specificity, and antigen heterogeneity. This systematic review evaluates microbial systems as innovative platforms to address these limitations through synthetic biology-driven approaches, with a focus on bridging preclinical advances to clinical implementation. Results Analysis of 389 peer-reviewed studies (2015–2025) reveals that engineered probiotic strains (e.g., Escherichia coli Nissle 1917) achieve selective tumor colonization while functioning as programmable factories for: 1. Synthetic antigen production and single-chain variable fragment (scFv) expression, 2. Costimulatory domain delivery enabling antigen-agnostic CAR-T activation, 3. Tumor microenvironment modulation via immunostimulatory chemokines. Microbial platforms demonstrate superior manufacturing economics (70–90% cost reduction vs. conventional methods) and enhance CAR-T functionality through epigenetic reprogramming by microbial metabolites (e.g., short-chain fatty acids). CRISPR/Cas-engineered genetic circuits further enable precise spatiotemporal control of therapeutic payloads. Conclusions Microbial systems represent transformative platforms for scalable, programmable CAR immunotherapy with significant potential for solid tumor targeting. Key barriers to clinical translation include biocontainment challenges, incomplete mechanistic understanding of tumor homing specificity, and safety validation requirements. Strategic integration of synthetic biology with microbial chassis offers a viable pathway toward accessible next-generation cancer therapies.

Abstract Background Toxoplasmosis, caused by Toxoplasma gondii ( T. gondii ), poses a significant global health threat with no commercial vaccine available. The parasite's egress process, which bridges its intracellular replication cycles and is critical for survival and dissemination, is tightly regulated by calcium. Notably, the T. gondii EF-hand domain-containing protein (Efhc) exhibits the highest Ca²⁺ binding affinity among its calcium-binding proteins. Methods CRISPR/Cas9 was used to generate a conditional knockout strain (TgEfhc3-C-AID). Phenotypic assays (plaque, intracellular proliferation, egress, invasion and murine virulence) were used to assess its impact on tachyzoites growth and development. Subsequently, TgEfhc3 antigenicity was analyzed using DNAstar software, Immunofluorescence assays and Western blots. Recombinant TgEfhc3 (rTgEfhc3) proteins, expressed in E. coli , was subcutaneously administrated to BALB/c mice to evaluate its protective efficacy against acute toxoplasmosis. Immune mechanisms induced by rTgEfhc3 were analyzed by measuring: serum IgG/IgG subclasses (IgG1, IgG2a) and splenic T cell cytokines (IL-4, IFN-γ, IL-10) by ELISA; the frequencies of CD4 + and CD8 + T cells by flow cytometry. Results Genetic deletion severely impaired tachyzoite proliferation, egress, and invasion, indicating its essentiality in T. gondii biology. Furthermore, recombinant TgEfhc3 was evaluated as a subunit vaccine in mice model, and was subsequently shown to protect against acute T. gondii infection. Immunization induced high levels of anti- T. gondii IgG and subclasses, enhanced Th1/Th2 cytokine production (IL-4, IFN-γ, IL-10) in splenic T lymphocytes, and stimulated robust CD4⁺ T cell proliferation. This elicited complex cellular and humoral immunity significantly prolonged survival time following acute T. gondii infection. Conclusions This study identifies that TgEfhc3 is crucial for T. gondii tachyzoite growth and development, providing new insights into infection mechanisms. Given the partial protective immunity conferred, TgEfhc3 warrants consideration as a component in future toxoplasmosis vaccine strategies.

Genome structural variants (SVs) comprise a sizable portion of functionally important genetic variation in all organisms; yet, many SVs evade discovery using short reads. While long-read sequencing can find the hidden SVs, the role of SVs in variation in organismal traits remains largely unclear. To address this gap, we investigate the molecular basis of 50 classical phenotypes in 11 Drosophila melanogaster strains using highly contiguous de novo genome assemblies generated with Oxford Nanopore long reads. These assemblies enabled the creation of a pangenome graph containing comprehensive, nucleotide-resolution maps of SVs, including complex rearrangements such as the interchromosomal inverted duplication Dp(2;4)eyD and large tandem duplications at the Bar locus. We uncovered new candidate causal mutations for 15 phenotypes and new molecular alleles for 2 mutations comprising tandem duplications, transposable element (TE) insertions, and indels. For example, we mapped the tarsal joint defect Ablp eyD to an 8 kb Roo retrotransposon insertion into an intergenic enhancer, a finding validated via CRISPR-Cas9. The wing vein phenotype plexus (px 1 ) was linked to a 1.5 kb partial tandem gene duplication, and the century-old Curved (c 1 ) wing phenotype was linked to a 7.5 kb DM412 retrotransposon inserted into the coding sequence of the muscle protein gene Strn-Mlck . We also unveiled 8 SV alleles of previously identified causal genes, including previously uncharacterized SVs underlying the extensively studied white and yellow phenotypes. Overall, 67.4% of the genes causing phenotypic changes harbored candidate SVs over 100 bp, whereas only 28% is expected based on euchromatic SVs. Our data, based on the 50 Drosophila phenotypes, 44 of which are strongly deleterious, suggests a disproportionately larger contribution of SVs to deleterious changes in visible phenotypes in Drosophila .

Neutrophils, the first cells to arrive at the site of inflammation, are rather short-lived cells and thus have to be constantly replenished. During neutrophil development, vesicle dynamics need to be fine-tuned and impaired vesicle trafficking has been linked to failure in neutrophil maturation. Here, we characterized the role of VPS18 as a central core component of CORVET & HOPS tethering complexes for neutrophil development. Using CRISPR/Cas9-engineered Hoxb8 cells with heterozygous mutations in Vps18 , we found that VPS18 deficiency interfered with neutrophil development due to tethering complex instability. As a result, vesicle dynamics were impaired with a strong increase in LC3-II and p62 levels, indicating autophagosome accumulation and reduced autophagic flux. With transmission electron microscopy, we verified the increase in autophagosomes and also found irregularly shaped vesicular structures in Vps18 mutants. Subsequently, Vps18 mutant neutrophil progenitors underwent premature apoptosis. We described a novel patient with a heterozygous stop-gain mutation in VPS18 suffering from neutropenia and recurrent infections. To verify our findings in the human system, we used human induced pluripotent stem cells (iPSCs). Upon differentiation into neutrophils, loss of VPS18 resulted in an almost complete absence of iPSC-derived developing neutrophils. Heterozygous VPS18 mutant and patient mutation-harboring iPSCs were characterized by strongly reduced numbers of developing neutrophils. Zebrafish larvae with heterozygous mutations in vps18 were also characterized by significantly reduced neutrophil numbers. This study shows the pivotal impact of VPS18 for adequate vesicle dynamics during neutrophil development which might be relevant in the context of vesicle trafficking during granulopoiesis and congenital neutropenia.

Abstract Hereditary persistence of fetal hemoglobin (HPFH) represents a clinically proven therapeutic model for β-hemoglobinopathies, where in persistent γ-globin (HBG1/HBG2) production compensates for impaired β-globin synthesis. To capitalize on this naturally occurring mechanism, we engineered a precise Clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) strategy targeting HBG regulatory elements to therapeutically reactivate fetal hemoglobin. Through optimized Cas9: sgRNA ribonucleoprotein (RNP) delivery, we implemented a dual-editing approach: (1) B-cell lymphoma/leukemia 11A (BCL11A) binding site disruption to alleviate transcriptional repression and (2) HPFH-associated deletion recapitulation. This strategy achieved robust fetal hemoglobin (HbF) reactivation in multiple experimental systems, including K562 erythroleukemia cells and primary hematopoietic stem/progenitor cell (HSPC)-derived erythroid cultures. The successful translation of this approach could provide a transformative treatment paradigm for β-thalassemia and sickle cell disease patients.

Abstract Many common diseases have a polygenic architecture. The responsible alleles are thought to mediate risk by disturbing gene regulation in most cases, however, the precise mechanisms have been elucidated only for a few. Here, we investigated the 16q23.3 genomic locus, which genome-wide significantly associates with coronary artery disease, a globally leading cause of death caused by accumulation of lipid-rich inflammatory plaques in the arterial wall. The locus harbors CDH13, whose mRNA and protein we found to be suppressed in atherosclerotic human and mouse arteries. Loss-of-function(LoF) variants of CDH13 were associated with detrimental cardiovascular phenotypes in the UK Biobank. Its knock-out increased plaque-sizes in Cdh13 -/- / Apoe -/- mice compared to Apoe -/- mice on a Western diet. After establishing an atheroprotective role of CDH13 , we studied its regulation. Integration of population genomic and transcriptomic datasets by GWAS-eQTL colocalization analysis identified CDH13 and four long non-coding RNAs (lncRNAs) as candidate causal genes at the 16q23.3 locus. dCas13-mediated RNA immunoprecipitation revealed that the lncRNA CDH13-AS2 binds to CDH13 mRNA in human endothelial cells (ECs). Its CRISPR/Cas9-based knockout in ECs was atherogenic, whereas dCas9-based transcriptional activation (CRISPRa) of CDH13-AS2 was atheroprotective; effects that were found to be mediated by the stability of CDH13 mRNA. To further understand how the CDH13-AS2 protects the mRNA we searched in silico and screened in vitro for microRNAs (miRNAs) that bind to CDH13 3’UTR. Indeed, four miRNAs, miR-19b-3p, miR-125b-2-3p, miR-433-3p, and miR-7b-5p, were found experimentally to accelerate CDH13 mRNA degradation, an effect that was neutralized by CRISPRa of CDH13-AS2 . Taken together, our study demonstrates an interplay of miRNAs, lncRNAs, and mRNA, which modulates the abundance of an atheroprotective protein in endothelial cells, which may offer a new therapeutic target for coronary artery disease.

Abstract Climate change is becoming a great challenge to sustainable crop production in sub-Saharan Africa. Conventional control methods are becoming ineffective against high-risk pests such as Spodoptera frugiperda , Bactrocera dorsalis , and Maruca vitrata . To overcome such challenges, we created an AI-CRISPR approach that combines real-time insect monitoring, climate-adaptive gene targeting, and field-tested suppression measures.The study utilised climatic modeling, host-pest phenology mapping, and population genomics to identify stable gene targets in life stages and climatic stress conditions. CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9) constructs were developed to break the fundamental gene controlling reproduction (vitellogenin), host detection (odorant-binding proteins), and thermotolerance, enhancing detection of pests by 88 percent and decreasing the use of pesticides by 35 percent.Simulated climate stress knockouts on specific traits mitigated oviposition of S. frugiperda by 72%, B. dorsalis fruit infestation by 65% and M. vitrata larval mortality by 81%. In Abuja and Makurdi, field trials demonstrated that population reduction of pests was at 83.4, 75.1, and 697.7 percent, respectively, and this resulted in an increase in the yield of crops by 28 percent and also a reduction of 40 percent in post-harvest losses. AI models forecast that the resurgence of the pest will occur by 61 percent after 2040–2050.The research constructed an AI-CRISPR-IPM pipeline, which can bolster climate resilience, diminish chemical reliance, and fit into local agriculture frameworks, providing an environmentally viable answer to African food security in a world that is warming.

SUMMARY Extracellular alkalinization has long been recognized as a hallmark of plant cell-surface receptor activation, including during pattern-triggered immunity (PTI); yet the mechanisms driving elicitor-induced alkalinization and its role in immune signaling remain unclear. Here, we demonstrate that inhibition of autoinhibited H + -ATPases (AHAs) is required for elicitor-induced extracellular alkalinization. This alkalinization is essential for immune signaling mediated by diverse plasma membrane-localized receptor kinases (RKs) through modulation of ligand-receptor interactions. Notably, RKs transduce elicitor-triggered signaling via BOTRYTIS-INDUCED KINASE 1 (BIK1), which inhibits AHA activity by disrupting AHA-GENERAL REGULATORY FACTOR (GRF) interactions through a conserved phosphorylation event. Interestingly, this pathway is crucial for cell wall damage (CWD) responses involving the RK MALE DISCOVERER 1-INTERACTING RECEPTOR LIKE KINASE 2 (MIK2) and its ligand, SERINE RICH ENDOGENOUS PEPTIDE 18 (SCOOP18). Our findings reveal a conserved phospho-regulatory pathway that governs extracellular alkalinization to coordinate plant immune signaling, offering new insights into plant stress resilience.