Abstract Electronic biosensors offer a compact, integrable platform for real-time biomolecular detection, yet their performance is often limited by charge screening in physiological environments. Here, we report a porous ZnO/Al2O3 biosensor with grain boundary-engineered nanostructures that enables attomolar-level (0.5 aM) detection of microRNAs (miRNAs). The porous architecture forms spontaneously during thermal annealing through Zn2+ diffusion toward the Al2O3 layer along the ZnO grain boundaries and through lateral surface diffusion, generating localized porosity at structurally defined sites. These regions disrupt charge neutrality and introduce energy barriers, thereby enhancing surface potential shifts upon miRNA hybridization. The biosensor maintains high sensitivity in high-ionic-strength buffers, indicating effective suppression of Debye screening. Using the CRISPR-Cas13a system, we functionalized the surface with a crRNA targeting miR-17, thereby achieving sequence-specific detection and demonstrating compatibility with programmable molecular recognition. This grain boundary-guided nanostructuring strategy establishes a scalable route to engineering biosensors with localized sensitivity and target selectivity. Our approach provides a platform for ultrasensitive nucleic acid detection and offers strong potential for integration into miniaturized diagnostic systems.

We determined the potential of CRISPR/Cas13 technology as a therapeutic approach for centronuclear myopathies (CNMs) by reducing the expression of a single protein, DNM2. CNMs are severe congenital rare muscle disorders that result in muscle hypotrophy and weakness, with no cure. CNMs frequently result from mutations in either BIN1 , MTM1 , or DNM2 genes, with DNM2 being a key GTPase that plays a pivotal role in muscle membrane interactions with MTM1 and BIN1. Previous studies indicate that reducing DNM2 transcript expression by half could correct CNM phenotypes regardless the genetic forms, paving the way for a broad-spectrum CNM-therapy. We evaluated CRISPR/Cas13X.1-mediated DNM2 transcript knockdown, as a therapeutic application in a unique naturally-occurring canine CNM model harboring the DNM2 R465W /+ mutation, the most frequent pathogenic variant in patients. We show that in vivo intramuscular AAV-mediated CRISPR/Cas13X.1 injections, led to a reduction in DNM2 transcript and protein levels at one and two months post-treatment. Our results demonstrate the feasibility of CRISPR/Cas13-based therapy for CNM in a large animal model, paving the way for advancing this approach towards clinical trials.

Achieving scalable and sustainable production of cultivated meat hinges on developing robust livestock muscle and fat cell lines that can proliferate and differentiate effectively, while meeting regulatory standards and consumer expectations. In this study, bovine satellite cells (BSCs) were immortalized using CRISPR/Cas9 to knockdown PTEN (phosphatase and tensin homolog), TP53 (cellular tumor antigen) and SMAD4 (SMAD [Sma and Mad proteins] family member 4). The resulting cell line, named “CriBSC2,” exhibited consistent growth, maintained muscle cell characteristics, and successfully differentiated into multinucleated myotubes after more than 150 cell divisions. In contrast, another cell line, “CriBSC1,” achieved immortalization with TP53 knockout alone but lacked differentiation capacity. CriBSC2 were further cultured on gelatin scaffolds to evaluate their anchorage-dependent responses, laying the groundwork for their potential application in tissue engineering for cellular agriculture. Ultimately, CriBSC2 cells demonstrate suitable proliferation and differentiation capabilities crucial for advancing cellular agriculture and future food technologies.

Relapsed and refractory T cell malignancies are associated with poor clinical outcomes. Autologous sources of αβT cells have been employed for chimeric antigen receptor (CAR) therapies to eliminate the potential for graft-vs-host disease (GvHD). However, the application of CAR-T therapy for T-ALL has been hindered by an inability to obtain sufficient healthy αβT cells from patients combined with fratricide due to concurrent antigen expression on normal T cells. Here, we genetically engineered polyclonal γδT cells, which do not cause GvHD, as an allogeneic source for cancer immunotherapy targeting the pancancer antigen CD38. Utilizing a novel expansion protocol in combination with CRISPR/AAV gene editing, we developed CD38KO/CD38-CAR polyclonal γδT cells that target T-ALL. Our editing strategy enabled site-directed, on-target insertion of the CD38-CAR transgene into the CD38 locus, with no evidence of significant random CAR DNA integration (as commonly seen with lentiviral CAR transduction) or chromatin abnormalities resulting from CRISPR editing. This enhanced targeting effectively mitigated fratricide through simultaneous CD38 disruption and CAR expression. We demonstrated the efficacy of the CD38KO/CD38-CAR γδT cells in vitro across multiple patient-derived T-ALL samples collected at baseline and relapse. In vivo, a single injection of CD38KO/CD38-CAR γδT cells without exogenous cytokine support resulted in potent anti-leukemic efficacy. Fratricide-resistant CD38KO/CD38-CAR polyclonal γδT cells thus represent a promising off-the-shelf therapeutic platform for T cell malignancies and other CD38-expressing cancers. Key Points Hybrid pan-γδTCR antibody/mbIL21-41BBL feeder expansion yields high-purity, polyclonal γδT cells suitable for CRISPR/AAV editing. On-target CD38-CAR knock-in with simultaneous CD38 knockout prevents fratricide and enables potent T-ALL killing in vitro and in vivo.

The CRISPR-Cas9 system is a powerful genome editing tool capable of precisely recognizing and cleaving specific DNA sequences, and has been extensively investigated as a strategy for correcting mutations associated with genetic diseases and cancer. However, conventional CRISPR genome engineering often fail to discriminate single-nucleotide mutations from wild-type alleles when the mutation is located outside the protospacer adjacent motif (PAM) sequence. To address this limitation, we developed a RNA engineering approach for designing near-complementary single guide RNA (sgRNA) that contain intentional mismatches within the seed region of the sgRNA. Single molecule kinetic analyses showed that the near-complementary sgRNA selectively reduces the binding affinity of CRISPR ribonucleoprotein complex by via differentiated increment in the dissociation rates to the wild-type target DNA compared to the mutant allele. The engineered kinetic characteristics of near-complementary sgRNAs enable highly specific genome editing of single-base mutations without reliance on PAM proximity. We demonstrate the application of the strategy to the a cancer-specific single-nucleotide G228A (-124C > T) mutation in the TERT promoter, frequently found in glioblastomas and other tumors, that does not generate a canonical PAM sequence. Our near-complementary sgRNA successfully induced selective editing of the mutant allele while sparing the wild-type sequence. Furthermore, single-molecule fluorescence resonance energy transfer (smFRET) analyss revealed distinct differences in binding kinetics between mutant and wild-type DNA, providing kinetic insight into the discrimination process. We conclude that the near-complementary sgRNA CRISPR editing strategy facilitates precise PAM-independent targeting of single-nucleotide mutations without protein engineering and offers a molecular framework for expanding the specificity and applicability of CRISPR-based genome and epigenome editing technologies.

ABSTRACT Organic acids such as fumaric acid are widely used in the food and beverage industry as acidulants and preservatives, while also serving as versatile precursors for industrially relevant compounds. Fumaric acid is still predominantly produced through petroleum-derived processes. To enhance production efficiency and diversify supply, we are engineering Kluyveromyces marxianus as a biosynthetic platform from renewable feedstocks. In previous work, we have established K. marxianus Y-1190 as a host for lactose valorization based on its high growth rate on lactose and its tolerance for acid conditions. Here, we establish a trifunctional genome-wide library for K. marxianus using CRISPR activation, interference, and deletion to allow identification of gene expression perturbations that enhance tolerance to fumaric acid. We determined that deletion of ATP7 , encoding a subunit of the mitochondrial F 1 F 0 ATP synthase, and overexpression of QDR2 and QDR3 , two previously uncharacterized members of the 12-spanner H⁺ antiporter (DHA1) family in K. marxianus, can enhance fumaric acid tolerance. We also found that integrated overexpression of both QDR2 and QDR3 in a Δ FUM1 background strain improved titers of fumaric acid production from 0.26 g L −1 to 2.16 g L −1 . Together, these results highlight roles for membrane transport and mitochondrial function in enabling fumaric acid tolerance and production in K. marxianus . HIGHLIGHTS - Trifunctional CRISPR-AID enables gene activation, interference & deletion in K. marxianus . - Genome-wide CRISPR-AID screen identifies guides conferring fumaric acid tolerance. - verexpressing of QDR2 and QDR3 increases fumaric acid tolerance and production. - Deletion of mitochondrial gene ATP7 significantly improves fumaric acid tolerance. - Fermentation of QDR2 and QDR3 overexpression strain yields 2.16 g L⁻¹ fumaric acid.

Expanding the range of Protospacer Adjacent Motifs (PAMs) recognized by CRISPR-Cas9 is essential for broadening genome-editing applications. Here, we combine molecular dynamics simulations with graph-theory and centrality analyses to dissect the principles of PAM recognition in three Cas9 variants - VQR, VRER, and EQR - that target non-canonical PAMs. We show that efficient recognition is not dictated solely by direct contacts between PAM-interacting residues and DNA, but also by a distal network that stabilizes the PAM-binding domain and preserves long-range communication with REC3, a hub that relays signals to the HNH nuclease. A key role emerges for the D1135V/E substitution, which enables stable DNA binding by K1107 and preserves key DNA phosphate locking interactions via S1109, securing stable PAM engagement. In contrast, variants carrying only R-to-Q substitutions at PAM-contacting residues, though predicted to enhance adenine recognition, destabilize the PAM-binding cleft, perturb REC3 dynamics, and disrupt allosteric coupling to HNH. Together, these findings establish that PAM recognition requires local stabilization, distal coupling, and entropic tuning, rather than a simple consequence of base-specific contacts. This framework provides guiding principles for engineering Cas9 variants with expanded PAM compatibility and improved editing efficiency.

CRISPR-Cas9 systems, adaptive defense mechanisms in bacteria and archaea, have been widely adopted as powerful gene editing tools, revolutionizing biological and medical research. In the first steps of CRISPR-Cas9 gene editing, the Cas9 protein, in complex with RNA, facilitates DNA melting and subsequent RNA-DNA hybrid formation, but the atomic-level mechanism of this fundamental process is not fully understood. Here, we present the results of long-timescale molecular dynamics simulations in which Cas9-RNA complexes bound to double-helical DNA and promoted the formation of RNA-DNA base pairs in a unidirectional, stepwise manner. Unexpectedly, we observed a direct role for the RNA in facilitating DNA melting events through a mechanism in which RNA bases intercalated within the DNA and promoted strand separation. In addition, breathing motions within the Cas9 DNA-binding cleft contributed to the sequential formation of RNA-DNA base pairs. These simulation results, obtained for two structurally distinct Cas9 proteins, together with supporting experimental work, suggest a novel RNA-dependent mechanism for DNA melting that may be conserved in other Cas proteins.

Abstract Background Colorectal cancer (CRC) exhibits limited responsiveness to immune-checkpoint blockade, necessitating further investigation. The intratumoral Treg/CD8⁺ T-cell ratio serves as a predictive biomarker for therapeutic efficacy. Here, we demonstrate that HSPB1 targeting reduces this ratio and confers therapeutic benefit in CRC. Methods Candidate genes were identified by integrative single-cell transcriptomics, TCGA and spatial transcriptomics, followed by survival analyses of TCGA cohorts. Functional interrogation was performed using CRISPR-Cas9 engineered knockout cell lines. Subcutaneous tumor models were established, and the immune microenvironment was characterized by multiparametric flow cytometry. Mechanistic validation was achieved through bulk RNA-seq and complementary functional assays. Results Single-cell profiling and TCGA WGCNA analyze identified HSPB1 as a putative determinant of the intratumoral Treg/CD8⁺ T-cell ratio, and survival analysis showed its prognostic relevance in CRC. Spatial transcriptomics revealed colocalization of HSPB1-expressing tumor cells with Tregs. Subcutaneous tumor models demonstrated that CRISPR-mediated HSPB1 deletion or pharmacologic inhibition markedly suppressed tumor growth and reprogrammed the Treg-dominated microenvironment. In vitro polarization assays confirmed that targeting HSPB1 selectively restrains Treg differentiation without affecting Th17. Integrated transcriptomic and functional studies further elucidated that HSPB1 orchestrates CCL20–CCR6 mediated Treg recruitment, thereby shaping the immunosuppressive milieu within colorectal tumors. Conclusions Targeting HSPB1 exerts dual anti-tumor effects: it directly suppresses neoplastic proliferation and simultaneously alleviates Treg-mediated immunosuppression within the tumor microenvironment.

The European Commission has proposed to amend the EU GMO regulation, exempting certain genetically modified plants generated with new genomic techniques (NGTs) from risk assessment. In the suggested lex specialis so-called “category 1 NGT plants” would be treated as equivalent to conventionally bred plants, if they meet threshold-based criteria, which limit the number and size of induced genetic changes. Here, we critically analyze the scientific validity of these thresholds and show that the proposal oversimplifies genetic complexity – disregarding the biological context, mutational bias, and functional consequences. The proposal’s central claim of equivalence between NGT1 plants and conventionally bred plants is thus scientifically unfounded. Many conceivable genetic modifications produced with NGTs – including those created with CRISPR prime editing and AI-assisted design – could be highly complex and exceed the capabilities of conventional breeding. Nevertheless, the regulatory proposal treats all possible genetic changes as equally likely and overlooks the purpose and function of genetic edits. By eliminating case-by-case risk assessment, the proposal creates a regulatory gap that allows complex and novel traits to bypass scrutiny – undermining the EU’s legally binding precautionary principle. In contrast, a risk-based regulatory approach is needed to ensure safe and future-proof oversight of NGT plants.

The CRISPR-Cas system is one of the most versatile and adaptive defense mechanisms in prokaryotes, facilitating sequence-specific identification and neutralization of invading genetic elements, such as bacteriophages and plasmids. Beyond their primary function in adaptive immunity, accumulating evidence indicates that CRISPR-Cas systems are intricately integrated into bacterial physiology and involve processes such as gene regulation, stress response, biofilm dynamics, quorum-sensing pathways, and virulence modulation. These functions underscore the multifaceted role of CRISPR-Cas in bacterial survival, persistence, and host-pathogen interactions. Moreover, the horizontal transfer and evolutionary diversification of CRISPR-Cas systems underscores their significance in shaping microbial communities and facilitating co-evolutionary interactions with phages. The translational potential of these systems extends well beyond microbial immunity and offers promising applications in microbiome engineering, antimicrobial development, and precision medicine. This review synthesizes the current knowledge on the regulatory and adaptive roles of CRISPR-Cas, highlighting their dual function as protectors of genomic integrity and modulators of host interactions.

Cellular stress responses are essential for maintaining homeostasis in the face of environmental or internal challenges. In the central nervous system, microglia serve as key stress sensors and immune responders, shaping neuroinflammatory processes and disease progression. However, the molecular programs engaged by distinct stressors and their impact on microglial viability remain incompletely understood. In this study, we used human induced pluripotent stem cell-derived microglia-like cells to investigate stress responses to amyloid beta (Aβ), a chronic Alzheimer’s disease–related stressor, and lipopolysaccharide (LPS), a classical acute inflammatory stimulus. Using single-cell RNA sequencing, we mapped the transcriptional programs activated by each condition and benchmarked these states against reference microglial datasets from mouse and human brains. In parallel, we performed a pooled CRISPR interference screen targeting Alzheimer’s disease-associated microglial genes to identify genetic determinants of microglial survival. We found that Aβ and LPS elicit partially overlapping but distinct transcriptional responses. Aβ induced more focused and disease-associated gene expression changes, while LPS triggered broad inflammatory activation and stronger cell death signatures. A subset of genes activated by stress overlapped with Alzheimer’s disease risk genes and with hits from the survival screen, suggesting that disease-associated microglial genes may contribute to stress adaptation and cellular fitness. These results demonstrate that iPSC-derived microglia-like cells can recapitulate in vivo–like stress-responsive states and offer a tractable platform to investigate genetic and environmental influences on microglial behavior. Together, our findings reveal transcriptional programs that link stress sensing, survival regulation, and Alzheimer’s disease–associated gene networks, providing a foundation for future efforts to enhance microglial resilience in neurodegenerative disease contexts.

Multiple epiphyseal dysplasia (MED), caused by mutations in MATN3, is a chondrodysplasia affecting the cartilage growth plate and is characterised by delayed epiphyseal ossification, short stature, and early onset osteoarthritis. Here we generated an in vitro human pluripotent stem cell (hPSC) model of cartilage growth-plate development to identify pathogenic mechanisms underlying MED. hPSCs were differentiated to chondrocytes via a mesenchymal intermediate, followed by TGFβ3+BMP2 induced chondrogenic pellet culture. MATN3-mutant hPSCs were generated by reprogramming MED patient PBMCs or by CRISPR-Cas9 gene editing to introduce a MATN3 mutation in a hESC line. RNAseq was used to assess chondrogenesis and identify MED pathogenic mechanisms. Transmission electron microscopy (TEM) was used to assess extracellular matrix assembly. The resultant hPSC-derived cartilage pellets displayed a typical cartilage morphology and strongly expressed cartilage matrix markers, e.g., collagen II and matrilin-3. Matrilin-3 protein was detected within both the matrix and cells of heterozygous mutant hPSC-cartilage pellets. RNAseq of mutant hPSC-cartilage pellets revealed significant enrichment for ‘ECM organisation’ and ‘cholesterol biosynthesis’ pathway genes as well as sightly increased expression of some unfolded protein response (UPR) marker genes. MATN3 mutant hPSC-derived cartilage pellets displayed abnormal matrix assembly, distended ER, accumulation of lipid droplets, and increased cholesterol content. Our model revealed mutant matrilin-3 induces cholesterol biosynthesis pathway upregulation and abnormal matrix assembly during MED pathogenesis. This study provides new insights into the molecular mechanisms underlying MED and highlights potential therapeutic targets.

Mutations in the MECP2 gene cause the severe neurological disorder Rett syndrome. A cluster of frameshift-causing C-terminal deletions (CTDs) lead to loss of ~100 amino acids at the C-terminus of the MeCP2 protein, and account for approximately 10% of RTT-causing mutations. The pathogenicity of C-terminal deletions (CTDs) is unexpected, as this C-terminal domain is non-essential in mice. Utilising databases of pathogenic and benign human MECP2 mutations, we find that some individuals with apparently typical CTDs do not exhibit Rett syndrome, confirming that C-terminal truncations are not intrinsically pathogenic. Using human DNA sequence data and mouse models, we demonstrate that pathogenicity results from a drastic reduction in MeCP2 levels and is determined by the presence of the short amino acid motif proline-proline-stop (-PPX) at the C-terminus, which results from a shift to the +2 reading frame. Individuals with CTDs that shift to the +1 frame avoid this motif and do not develop Rett syndrome. Mutating the stop codon of the PPX motif to tryptophan rescues MeCP2 expression and RTT-like phenotypes in a CTD mouse model. Finally, we demonstrate that an adenine base editor can efficiently introduce this tryptophan substitution in cultured cells. Overall, our findings uncover a simple and reliable prognostic distinction between benign and pathogenic CTDs and provide proof-of-concept for an editing strategy that potentially corrects all disease-causing CTD mutations.

ABSTRACT How cell contact initiates T-cell activation is uncertain. The local exclusion of the receptor-type protein tyrosine phosphatase CD45 at cell contacts is believed to trigger immune receptor signaling but this is yet to be observed for T cells interacting with authentic cellular targets. Here, quantitative imaging of T cells interacting with tumor cells presenting either native or clinically relevant bi-specific TCR ligands, revealed that they form multiple sub-micron sized ‘close contacts’ with their targets. The contacts were stabilised by the adhesion protein CD2, but efficient ligand detection required both CD2 and integrin ligation. CD45 was excluded from close contacts at the time of ZAP70 recruitment and signaling, but only partially (30− 40%). A single-cell, mass cytometric analysis showed that this change in kinase/phosphatase activity provoked strong T-cell activation and potent cytotoxicity via very small changes in signaling fluxes. Spatial stochastic simulations suggested that the proximal T-cell signaling network is optimised for efficient antigen discrimination in the setting of partial CD45 exclusion. Our work re-frames early T-cell activation as a process initiated by relatively subtle changes in kinase/phosphatase activity acting on small numbers of signaling effectors at minute cellular contacts.

ABSTRACT Fusobacterium nucleatum is a Gram-negative anaerobe associated with periodontitis and colorectal cancer. It secretes putrescine, a polyamine that promotes biofilm formation by oral co-colonizers and enhances the proliferation of cancer cells. However, the physiological importance of putrescine for F. nucleatum itself remains unexplored. Here, we show that putrescine biosynthesis, mediated by the ornithine decarboxylase gene oda , is essential for F. nucleatum viability. Deletion of oda was only possible when a functional copy was provided in trans, and CRISPR interference of oda expression resulted in complete growth arrest. The essentiality of oda was conserved across multiple subspecies. Supplementation with exogenous putrescine enabled the isolation of a conditional oda mutant whose growth was strictly putrescine-dependent. Putrescine depletion caused filamentation, membrane disruption, detergent hypersensitivity, and lysis in hypoosmotic conditions, indicating a critical role in maintaining cell envelope integrity. RNA sequencing revealed broad transcriptional remodeling under putrescine-limited conditions, including upregulation of genes involved in lipid metabolism, osmoprotection, and cell wall remodeling. Notably, oda transcript levels increased when putrescine was depleted, suggesting a negative feedback mechanism. These findings demonstrate that putrescine is not only an extracellular communal metabolite but is also vital for the cellular integrity and survival of F. nucleatum under anaerobic conditions. IMPORTANCE Fusobacterium nucleatum is a prominent member of the oral microbiota and has been linked to various human diseases, including periodontitis, preterm birth, and colorectal cancer. Despite its clinical significance, the metabolic requirements that support its growth and viability remain poorly understood. In this study, we identify the oda gene, which encodes ornithine decarboxylase, as essential for F. nucleatum survival due to its role in putrescine biosynthesis. We demonstrate that depletion of putrescine leads to severe growth and morphological defects, accompanied by widespread transcriptional changes. These findings reveal an underappreciated metabolic vulnerability and highlight the critical role of polyamine homeostasis in maintaining cellular integrity in this notorious anaerobe.

The CRISPR-Cas9 system has revolutionized genome engineering, but its clinical and research success hinges on the design of highly efficient and specific guide RNAs (gRNAs). This design process presents a complex multi-objective optimization challenge. Current computational approaches often rely on single-pass prediction models or require researchers to build bespoke, difficult-to-maintain scripting pipelines for iterative discovery workflows. Here, we introduce GeneForgeLang (GFL), a novel domain-specific language (DSL) designed to declaratively specify and orchestrate advanced AI-driven workflows in genomics. We demonstrate GFL's capabilities by applying its high-level guided_discovery abstraction to the problem of optimizing gRNAs for the human tumor suppressor gene TP53. Our workflow, defined in a single, readable GFL script, autonomously orchestrates an iterative cycle of candidate generation, predictive evaluation using deep learning models, and active learning-based selection. In just 5 cycles, the system efficiently evaluated 100 informative candidates, converging on solutions with near-optimal predicted performance scores (top score: 0.9859). GFL represents a new paradigm for enhancing the reproducibility, composability, and speed of computational research in the life sciences.

Stabilisation of Cyclin D2 is the underlying cause of a range of neurodevelopmental disorders, characterised by megalencephaly, cortical migration defects and overgrowth. Intracellular CCND2 is vital for mTOR pathway signalling, with inhibition of mTOR resulting in CCND2 phosphorylation and degradation by the ubiquitin proteasome system. Mutations at the regulatory c-terminus of CCND2, and in proteins that regulate mTOR such as PTEN, PIK3CA, AKT3 and TSC1/2, result in CCND2-stabilisation and overgrowth. To determine the molecular and cellular mechanisms underpinning the neurodevelopmental defects observed in Megalencephaly-Polymicrogyria-Polydactyly-Hydrocephalus (MPPH) syndrome, we generated human induced pluripotent stem cell (iPSC) derived models of CCND2-associated disease. Using CRISPR-Cas9 we generated lines containing either a pathogenic CCND2 variant (c.814G>T, p.Glu272Ter) or frameshift variants in the final exon of CCND2 , all of which truncate CCND2 before the critical Thr-280 residue, required for its phosphorylation and degradation. We observed truncating frameshift variants do not result in CCND2 stabilization, whereas the single nucleotide c.814G>T, p.Glu272Ter substitution does, mimicking the effect seen in MPPH patients. Differentiation into human cortical spheroids (hCS) revealed all CCND2-truncating lines continued to express PAX6 beyond the neural progenitor (NP) expansion phase. Furthermore, both the homozygous and heterozygous p.Glu272Ter hCS failed to produce mature Tbr-1 expressing neurons, while some expression was observed in the frameshift hCS, highlighting differences in neurogenesis between frameshift and nonsense lines. Despite all lines truncating CCND2 and removing Thr-280, our data implies that frameshift truncations do not stabilise CCND2. In comparison, truncation of CCND2 through introduction of a single nucleotide nonsense variant results in CCND2 stabilisation, mimicking MPPH.

Abstract Background Pompe disease is an autosomal recessive lysosomal storage disorder caused by mutations in the GAA gene, leading to acid alpha-glucosidase deficiency and pathological glycogen accumulation, primarily in cardiac and skeletal muscle. While enzyme replacement therapy (ERT) has improved clinical outcomes, its limited efficacy especially in skeletal muscle underscores the need for improved disease models and novel therapeutic strategies. Induced pluripotent stem cells (iPSCs) from Pompe patients have facilitated mechanistic studies; however, their utility is restricted by limited patient sample availability. Methods To address this limitation, we employed CRISPR-Cas9 genome editing to disrupt GAA in a well-characterized human embryonic stem cell (hESC) line, BJNhem20, thereby generating a Pompe disease model independent of patient material. Results The edited hESC line exhibited markedly reduced GAA enzymatic activity while maintaining pluripotency and trilineage differentiation potential. Upon directed differentiation, cardiomyocytes displayed pronounced lysosomal accumulation and increased glycogen storage, whereas skeletal myotubes exhibited elevated cell death and a marginal increase in glycogen content. Conclusions These findings demonstrate that genome-edited hESC for Pompe disease can recapitulate key pathological features, providing a robust and scalable platform for disease modelling and therapeutic screening. This approach offers a valuable alternative to patient-derived iPSCs for studying rare genetic disorders and for the development of targeted interventions.

To systematically identify causal genetic mechanisms that confer risk for coronary artery disease (CAD) in GWAS loci, we mapped genome-wide variant-to-enhancer-to-gene (V2E2G) links in vascular smooth muscle cells (SMC). Enhancers identified by active chromatin features, and further prioritized by base-resolution deep learning models of chromatin accessibility in 108 CAD loci, were studied with CRISPRi targeting and Direct-Capture Targeted Perturb-seq (DC-TAP-seq) evaluation of 470 genes. Seventy-six V2E2G links were identified for 59 candidate CAD genes representing gene programs including epithelial-mesenchymal transformation, ubiquitination, and protein folding as well as BMP and TGFB signaling. Similar methods employed with an independent focused screen targeting one candidate locus at 9p21.3 identified 10 enhancers regulating expression of multiple genes at this location. Detailed molecular studies revealed that two enhancers mediating transcription factor binding and transcriptional regulation contribute to ancestry-specific and sex-specific risk for CAD and the surrogate biomarker vascular calcification. Together, these studies advance our identification of GWAS CAD V2E2G links across the genome, and specific mechanisms of risk at the complex 9p21.3 locus.

Introduction Sodium-glucose cotransporter 2 (SGLT2) is a key mediator of renal glucose reabsorption. Its pharmacological inhibition exerts cardio- and reno-protective benefits. Despite widespread clinical interest, reliable detection of SGLT2 protein remains challenging due to concerns regarding the specificity of available antibodies. Methods This study assessed the specificity of eight commercially available anti-SGLT2 antibodies by immunohistochemistry and Western blotting. Genetically engineered Sglt2 -deficient mice and rats, generated via clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology, were employed as definitive negative controls. Additionally, human kidney tissues, including renal cell carcinoma samples, were analyzed. Results Among the antibodies tested, few exhibited robust specificity, characterized by substantial immunostaining with minimal background in wild-type kidney tissues and complete absence of staining in Sglt2 -deficient samples. In renal cell carcinoma samples, a validated antibody detected SGLT2 immunostaining in proximal tubules of non-tumor regions but not in tumor areas. Subcellular localization studies revealed that SGLT2 was enriched within proximal tubular microvilli, partially overlapping with its co-factor PDZK1IP1 (MAP17). LRP2 (megalin) and NHE3 were placed at the microvillar base and did not colocalize with SGLT2. Western blotting identified a specific SGLT2 band at approximately 55 kDa in kidney lysates using several antibodies under optimized procedures. This band was shifted to approximately 45 kDa after enzymatic removal of N-linked glycans. One antibody detected a weak band at the same molecular mass even in kidney lysates from Sglt2 -deficient rodents. Conclusions Considerable variability exists in the specificity of commercially available anti-SGLT2 antibodies. Only a limited number of antibodies are suitable for reliable detection of SGLT2 in rodent and human samples. Rigorous antibody characterization, including the use of knockout controls and optimized experimental conditions, is essential to ensure reproducibility and prevent misinterpretation in studies investigating the biological and pathophysiological roles of SGLT2.

Selecting appropriate experimental systems is crucial in cancer research, where factors such as model relevance, cost, and resource availability guide decisions. A detailed understanding of the strengths and limitations of each model helps ensure their optimal use. We recently developed a human lung squamous cell carcinoma (LUSC) model using genetically engineered human bronchial epithelial cells (hBECs). These were studied through organotypic air-liquid interface (ALI) cultures and standard in vitro assays, including proliferation, invasion, and anchorage-independent growth. However, we did not evaluate whether the same mutant hBECs behaved similarly in vivo, or if in vivo models offered distinct advantages. To address this, we conducted a comparative phenotypic analysis of mutant hBECs derived from the same donor in both ALI cultures and xenografts in immunocompromised mice. Both models followed a similar oncogenic trajectory, involving squamous differentiation and activation of Nrf2 and PI3K/Akt pathways, characteristic of the classical LUSC subtype. However, some transcriptomic differences related to an increase in microtubule formation and cell motility in xenografts emerged. Additionally, xenograft gene expression profiles more closely matched classical LUSC tumours than ALI cultures. Importantly, we observed spontaneous squamous differentiation in the absence of SOX2 overexpression and detected selection for NOTCH1 mutations in specific in vivo mutants. Truncation of NOTCH1 promoted squamous differentiation and suppressed mucociliary features in ALI cultures, underscoring its role as a potential LUSC driver. In summary, mutant hBECs in vitro and in vivo showed largely consistent phenotypes, validating both systems. However, in vivo models can enable the unbiased discovery of new genetic LUSC driver genes. This highlights the complementary value of integrating both model types in LUSC research.

Bacterial defense systems present considerable barriers to both phage infection and plasmid transformation. These systems target mobile genetic elements, limiting the efficacy of bacteriophage-based therapies and restricting genetic engineering applications. Here, we employ a de-novo protein design approach to generate proteins that bind and inhibit bacterial defense systems. We show that our synthetically designed proteins block defense, and that phages engineered to encode the synthetic proteins can replicate in cells that express the respective defense system. We further demonstrate that a single phage could be engineered with multiple anti-defense proteins, yielding improved infectivity in bacterial strains carrying multiple defense systems. Finally, we show that plasmids that express synthetic anti-defense proteins can be introduced into bacteria that naturally restrict plasmid transformation. Our approach can broaden host ranges of therapeutic phages and can improve genetic engineering efficiency in strains that are typically difficult to transform.

Abstract BCR signal dependency is a hallmark of diffuse large B-cell lymphoma (DLBCL) and other B-cell lymphoid malignancies originating from germinal centers. Chronic-active BCR signaling, typical for the more aggressive activated B-cell subtype (ABC) of DLBCLs, is often attributed to activating mutations within the BCR signaling cascade and continuous stimulation of the BCR by autoantigens. In certain ABC-DLBCLs, the BCR forms an intracellular multiprotein supercomplex with TLR9 and MYD88, which generates signals from endolysosomes. However, it is not clear whether the internalization of BCR is required for sustained signaling, nor have the mechanisms responsible for BCR trafficking been defined. A detailed and mechanistic characterization of receptor trafficking and its consequences is crucial for elucidating new therapeutic targets. To address these questions, we developed DLBCL cell models with modified ovalbumin (OVA)-specific hypervariable regions (HVRs) in the BCRs using CRISPR-Cas9 technology. Modified BCRs were incapable of binding self-antigens, while still responding in a controlled fashion to stimulation with ovalbumin. Using these genetic models, we demonstrated that autoantigens drive a complex BCR-dependent signaling program and facilitate the assembly of the intracellular BCR-TLR9-IκB complex, promoting NFκB pathway activation. Furthermore, we showed that the binding of autoantigens to the BCR leads to the internalization of the BCR-autoantigen complex via clathrin-mediated endocytosis (CME). Using genetic models with inducible inhibition of this endocytic pathway, we found that BCR internalization is essential for the oncogenic activation of BCR-dependent signaling pathways and the formation of the BCR-TLR9-IκB complex in autoantigen-dependent ABC-DLBCL cells. Finally, CME inhibition with dynamin-2 antagonists, such as phenothiazine derivatives, reduces BCR signaling, cell viability, and synergizes with SYK and PI3Kδ inhibitors. Since phenothiazines have well-defined safety and pharmacokinetic profiles, our data provide a framework for the rational design of clinical trials employing these drugs in the autoantigen-dependent subset of DLBCL.

Duckweeds ( Lemnaceae ) have excellent potential for fundamental and applied research due to ease of cultivation, small size, and continuous fast clonal growth. However, their usage as model organisms and platforms for biotechnological applications is often limited by the lack of universal genetic manipulation methods necessary for transgene expression, gene editing, and other methods to modify gene expression. To identify suitable strains for genetic manipulation of the giant duckweed, Spirodela polyrhiza, we screened several genotypes for callus induction and regeneration and established genetic transformation. We have identified SP162 to be amenable to Agrobacterium -mediated transformation via tissue culture. The procedure is robust and reproducible across laboratories, allowing stable expression of different reporter genes and selectable markers, enabling CRISPR/Cas9-mediated genome editing. In addition, due to a weak small RNA-based silencing response, S. polyrhiza sustains prolonged periods of transgene activity in transient expression assays. To promote duckweed research and encourage the adoption of S. polyrhiza , we have made SP162 (ID#: 5676 ) and its genome publicly available and provide here detailed procedures for its cultivation and transformation. Furthermore, we created a web server to explore its genome, retrieve gene sequences, and implemented orthologous gene search and a gRNA design function for diverse CRISPR/Cas-based applications ( https://agxu.uni-mainz.de/SP162/ ).