Summary CRISPR-Cas systems confer prokaryotic adaptive immunity by integrating foreign DNA (prespacers) into host arrays. Type II-A systems employ Cas9 for protospacer adjacent motif (PAM) recognition and coordinate with Csn2 and the Cas1-Cas2 integrase during spacer acquisition, yet their structural basis remains unresolved. Here, we report cryo-EM structures of the Enterococcus faecalis Cas9-Csn2-Cas1-Cas2 supercomplex in apo and DNA-bound states. The apo-state structure (Cas9 2 -Csn2 8 -Cas1 8 -Cas2 4 ) adopts a resting conformation, with Cas9 locked in a nuclease-inactive state and Cas1-Cas2 sterically blocked from prespacer loading. Upon DNA engagement, Cas9 undergoes a conformational transition, forming a prespacer catching complex that threads the DNA through Csn2’s central channel. This architecture enables Cas9 to interrogate the PAM sequence while sliding along the DNA, with Cas9 and Csn2 jointly define a 30-bp DNA segment which matches the prespacer length. Subsequent dissociation of Cas9 triggers a structural reconfiguration of the Csn2-Cas1-Cas2 assembly. The PAM-proximal DNA becomes accessible, and Cas1-Cas2 relocates to bind to the exposed DNA, enabling further prespacer processing and directional integration. These findings reveal how Cas9 collaborates with Csn2 and Cas1-Cas2 to couple PAM recognition with prespacer selection, resolving the dynamic structural transitions that ensure fidelity during type II-A CRISPR adaptation.

SUMMARY During CRISPR-Cas adaptation, prokaryotic cells become immunized by the insertion of foreign DNA fragments, termed spacers, into the host genome to serve as templates for RNA-guided immunity. Spacer acquisition relies on the Cas1-Cas2 integrase and accessory proteins like Cas4, which select DNA sequences flanked by the protospacer adjacent motif (PAM) and insert them into the CRISPR array. It has been shown that in type II-A systems selection of PAM-proximal prespacers is mediated by the effector nuclease Cas9, which forms a ‘supercomplex’ with the Cas1-Cas2 integrase and the Csn2 protein. However, the supercomplex structure and the role of the ring-like Csn2 protein remain unknown. Here, we present cryo-electron microscopy structures of the type II-A prespacer selection supercomplex in the DNA-scanning and two different PAM-bound configurations. Our study uncovers the mechanism of Cas9-mediated prespacer selection in type II-A CRISPR-Cas systems, and reveals the role of the accessory protein Csn2, which serves as a platform for the assembly of Cas9 and Cas1-Cas2 integrase on prespacer DNA, reminiscent of the sliding clamp in DNA replication. Repurposing of Cas9 by the CRISPR adaptation machinery for prespacer selection characterized here demonstrates Cas9 plasticity and expands our knowledge of the Cas9 biology.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of mortality worldwide. Proteins involved in lipid metabolism, such as the monoacylglycerol lipase Rv0183, play critical roles during both the active and dormant phases of Mtb and present novel targets for therapeutic intervention. Through high-throughput screening at the European Lead Factory, we identified a novel chemotype characterized by a hydroxypyrrolidine ring, which demonstrated potent inhibition of Rv0183 and promising results in whole cell bacterial studies. Subsequent co-crystallization studies of this chemotype with Rv0183 revealed non-covalent interactions within the lipase’s binding pocket, elucidating the inhibitory mechanism. Comparative analysis, augmented by AI-driven 3D-point-cloud approaches, distinguished Rv0183’s ligand-binding cavity from that of human monoacylglycerol lipase, implying the possibility for species-selective inhibition. This selectivity was further supported by molecular docking simulations which validated the experimental binding affinities and predicted strong, specific binding modes. Our study presents not only the structural basis for the inhibition of Rv0183 by these novel hydroxypyrrolidine-based inhibitors but also demonstrates the utility of integrating computational and empirical methods to achieve species-specific targeting. This approach could minimize off-target effects in humans, marking a significant step toward developing more effective antitubercular therapies. The potential to selectively inhibit Mtb in its dormant state could lead to treatments that prevent the persistence and resurgence of the disease, addressing a crucial gap in the fight against tuberculosis.

Multi-omics technologies allow for a detailed characterization of cell types and states across multiple omics layers, helping to identify features that differentiate biological conditions, such as chemical or CRISPR-based perturbations. However, current tools employing variational inference on single-cell datasets, including methods for paired and mosaic integration, transfer learning, and modality imputation, typically act as black boxes. This lack of interpretability makes it challenging to evaluate whether biological variation is preserved, which can compromise downstream analyses. Here, we introduce NetworkVI, a sparse deep generative model designed for the integration and interpretation of multimodal single-cell data. NetworkVI utilizes biological prior knowledge as an inductive bias, specifically it relies on gene-gene interactions inferred from topologically associated domains and structured ontologies like the Gene Ontology to aggregate gene embeddings to cell embeddings, enhancing the interpretability at the gene and subcellular level. While achieving state-of-the-art data integration, modality imputation, and cell label transfer via query-to-reference mapping benchmarks across bimodal and trimodal datasets, NetworkVI additionally excels in providing biologically meaningful modality- and cell type-specific interpretations. NetworkVI aids researchers in identifying associations between genes and biological processes and uncovers immune evasion mechanisms in a Perturb CITE-seq dataset of melanoma cells. NetworkVI will support researchers in interpreting cellular disease mechanisms, guiding biomarker discovery, and ultimately aiding the development of targeted therapies in large-scale single-cell multimodal atlases. NetworkVI is available at http://github.com/LArnoldt/networkVI .

Bacterial pathogens commonly become drug resistant via horizontal acquisition of antimicrobial resistance genes (ARGs), which are often encoded on mobile genetic elements (MGEs). Although bacterial defence systems are typically considered barriers to horizontal gene transfer (HGT), previous studies revealed that bacteria with more restriction-modification (RM) systems (the most abundant bacterial defences) frequently carry more MGEs. It was suggested that this counterintuitive relationship might result from stronger selection for RM systems when exposure to costly MGEs increases. Here, we test this hypothesis using a combination of modelling and bioinformatics analysis of >40,000 bacterial genomes to better understand how eco-evolutionary feedbacks between selection for RM and acquisition of MGEs shape bacterial genome evolution. Our model predicts negative associations between HGT and RM, but only if RM diversity is high. By contrast, at low RM diversity, eco-evolutionary feedbacks drive the emergence of positive associations between HGT and RM. Consistent with these predictions, we identified negative relationships between acquired ARG counts and RM counts across species but positive relationships within individual species. Collectively, our work helps to understand how RM systems shape patterns of HGT of ARGs, which may offer opportunities for targeted surveillance of strains at higher risk of horizontally acquiring novel drug resistance alleles.

SUMMARY Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify Cas1 and the Cas2/3 fusion protein from Pseudomonas aeruginosa . We determine multiple structures of the Cas1-2/3 complex at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Taken together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome. GRAPHICAL ABSTRACT HIGHLIGHTS - A positively charged channel on the Cas1-2/3 complex captures fragments of DNA - A loop in the RecA1 domain controls access to the Cas3 nuclease active site - Foreign DNA binding allosterically regulates access to additional DNA binding sites - Distortion of the CRISPR repeat sequence licenses complete foreign DNA integration

In Parkinson’s disease and other synucleinopathies, α-synuclein (α-Syn) misfolds and forms Ser 129 – phosphorylated aggregates (pSyn 129 ). The factors controlling this process are largely unknown. Here, we used arrayed CRISPR-mediated gene activation and ablation to discover new pSyn 129 modulators. Using quadruple-guide RNAs (qgRNAs) and Cas9, or an inactive Cas9 version fused to a synthetic transactivator, we ablated 2304 and activated 2428 human genes related to mitochondrial, trafficking and motility function in HEK293 cells. After exposure of cells to α-Syn fibrils, pSyn 129 signals were recorded by high-throughput fluorescent microscopy and aggregates were identified by image analysis. We found that pSyn 129 was increased by activating the mitochondrial protein OXR1, which decreased ATP levels and altered the mitochondrial membrane potential. Instead, pSyn 129 was reduced by ablation of the endoplasmic reticulum (ER)-associated protein EMC4, which enhanced ER-driven autophagic flux and lysosomal clearance. OXR1 activation preferentially modulated cellular reactions to fibrils derived from multiple system atrophy (MSA) patients, whereas EMC4 ablation broadly reduced pSyn 129 across diverse α-Syn polymorphs. These findings were confirmed in human iPSC-derived cortical and dopaminergic neurons, where OXR1 preferentially promoted somatic aggregation and EMC4 reduced both somatic and neuritic aggregates. These results uncover previously unrecognized roles for OXR1 and EMC4 in α-Syn aggregation, thereby broadening our mechanistic understanding of synucleinopathies.

Protein tyrosine kinases activate signaling pathways by catalyzing the phosphorylation of tyrosine residues in their substrates. Mounting evidence suggests that, in addition to recognizing phosphorylated tyrosine (pTyr) residues through specific phosphobinding modules, many protein kinases selectively recognize pTyr directly adjacent to the tyrosine residue they phosphorylate and catalyze the formation of twin pTyr-pTyr sites. Here, we demonstrate the importance of this phosphopriming-driven twin pTyr signaling in promoting cell cycle progression through the cell cycle-inhibitory protein p27 Kip1 . We identify, structurally resolve, and tune two distinct molecular determinants driving the selective recognition of pTyr directly N- and C-terminal to the target phospho-acceptor tyrosine site. We further show structural and biochemical conservation in this recognition, and identify cancer-associated alterations to these determinants that are unable to recognize phosphoprimed substrates. Finally, using an in vivo mouse model of leukemia we show that Bcr-Abl mutants unable to recognize phosphoprimed substrates paradoxically result in enhanced tumor development and progression. These data indicate that Bcr-Abl, like other proto-oncogenes such as Ras or Myc, engages both pro- and anti-oncogenic programs – but in the case of Bcr-Abl, this is accomplished through a mechanism involving traditional and phosphoprimed substrate recognition.

Single-guide RNA lentiviral infection with Cas9 protein electroporation (SLICE) enables CRISPR screening in primary cell types that require transient Cas9 expression yet is limited by scalability and robustness. Here, we introduce dual-guide RNA infection with Cas9 electroporation (DICE), which expresses two guides from the same lentiviral construct that target the same gene. In genome-wide screens, DICE outperformed SLICE in defining essential genes and modulators of PD-L1 expression in IFN gamma activated THP1 cells. Collectively, these data demonstrate that DICE can be utilized for reduced-scale CRISPR screens in cell types with transient Cas9 protein expression without sacrificing screening quality.

ABSTRACT Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP) 1 . Although the seven σ factors in E. coli have been extensively characterized 2 , Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ E ) whose precise roles are unknown, pointing to additional layers of regulatory potential 3 . Here we uncover an unprecedented mechanism of RNA-guided gene activation involving the coordinated action of σ E factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically-linked dCas12f and σ E homologs in E. coli using RIP-seq and ChIP-seq experiments, revealing systems that exhibited robust guide RNA enrichment and DNA target binding with a minimal 5ʹ-G target-adjacent motif (TAM). Recruitment of σ E was dependent on dCas12f and guide RNA (gRNA), suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σ E ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σ E complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription (RGT) that embodies natural features reminiscent of CRISPRa technology developed by humans 4,5 .

ABSTRACT RNA-guided proteins have emerged as critical transcriptional regulators in both natural and engineered biological systems by modulating RNA polymerase (RNAP) and its associated factors 1-5 . In bacteria, diverse clades of repurposed TnpB and CRISPR-associated proteins repress gene expression by blocking transcription initiation or elongation, enabling non-canonical modes of regulatory control and adaptive immunity 1,6,7 . Intriguingly, a distinct class of nuclease-dead Cas12f homologs (dCas12f) instead activates gene expression through its association with unique extracytoplasmic function sigma factors (σ E ) 8 , though the molecular basis has remained elusive. Here we reveal a novel mode of RNA-guided transcription initiation by determining cryo-electron microscopy structures of the dCas12f-σ E system from Flagellimonas taeanensis . We captured multiple conformational and compositional states, including the DNA-bound dCas12f-σ E -RNAP holoenzyme complex, revealing how RNA-guided DNA binding leads to σ E -RNAP recruitment and nascent mRNA synthesis at a precisely defined distance downstream of the R-loop. Rather than following the classical paradigm of σ E -dependent promoter recognition, these studies show that recognition of the −35 element is largely supplanted by CRISPR-Cas targeting, while the melted −10 element is stabilized through unusual stacking interactions rather than insertion into the typical recognition pocket. Collectively, this work provides high-resolution insights into an unexpected mechanism of RNA-guided transcription, expanding our understanding of bacterial gene regulation and opening new avenues for programmable transcriptional control.

ABSTRACT Oxytocin receptors (OTR) within the extended amygdala and nucleus accumbens have been implicated in modulating social behaviors, particularly following stress. The effects of OTR could be mediated by modulating the activity of pre-synaptic axon terminals or via post-synaptic neurons or glia. Using a viral-mediated CRISPR/Cas9 gene editing system in California mice ( Peromyscus californicus ), we selectively knocked down OTR in the anteromedial bed nucleus of the stria terminalis (BNST) or the nucleus accumbens (NAc) to examine their roles modulating social approach and vigilance behaviors. Knockdown of OTR in the BNST attenuated stress-induced decreases of social approach and increases of social vigilance behaviors in adult female California mice, similar to prior pharmacological studies. These effects were more prominent in the large arena social interaction where mice could control proximity to a social target with a barrier (wire cage). In a small arena interaction test where focal mice freely interacted with target mice, effects of BNST OTR knockdown were muted. This suggests that within the BNST OTR are more important for modulating behavioral responses to more distal stimuli versus more proximal social contexts. In mice with OTR knockdown in the NAc, few behavioral changes were observed which is consistent with previous findings of the importance of presynaptic OTR, which were unaffected by our gene editing strategy, driving social approach behaviors in the NAc. Interestingly, BNST OTR knockdown increased exploratory behavior toward a non-social stimulus after stress, pointing to a potentially broader role for BNST OTR function. Our findings highlight the region- and context-specific functions of OTR in social behavior and the advantages of using a selective gene-editing tool to dissect the neural circuits that influence social and stress-related responses.

Abstract The fall armyworm (FAW), Spodoptera frugiperda , is an invasive and polyphagous pest that has become a major threat to tropical crops due to its rapid dissemination, broad host range, and increasing insecticide resistance. This research approaches the molecular basis of resistance by combining comparative transcriptomics with RNA interference (RNAi) to discover and functionally validate critical detoxification genes. We identified significant up-regulation of cytochrome P450 monooxygenases (CYP321A1 and CYP9A4), glutathione S-transferases (GSTE6 and GSTE2) and carboxylesterase (CCE2) in resistant populations. Suppression of these genes through RNAi resulted in greater susceptibility to insecticides and lower larval survival. Delivery of the chitosan nanoparticle using dsRNA also improved stability and knockdown in a field context. Phylogenetic analysis revealed that these resistance genes are well conserved among the most important lepidopteran pests, indicating potential for a broader efficacy of RNAi as a pest management strategy. It may establish RNAi- and CRISPR-based molecular biocontrol systems in tropical IPMs, with significant benefits for sustainable agriculture and biodiversity conservation.

Gene editing technologies such as CRISPR/Cas9 have revolutionized functional genomics, yet their application in marine fish cell lines remains limited by inefficient delivery. This study compares three delivery strategies—electroporation, lipid nanoparticles (LNPs), and magnetofection using gelatin-coated superparamagnetic iron oxide nanoparticles (SPIONs)—for CRISPR/Cas9-mediated editing of the ifi27l2a gene in DLB-1 and SaB-1 cell lines. We evaluated transfection and editing efficiency, intracellular Cas9 localization, and genomic stability of the target locus. Electroporation achieved up to 95% editing in SaB-1 under optimized conditions, but only 30% in DLB-1, which exhibited locus-specific ge-nomic rearrangements. Diversa LNPs enabled intracellular delivery and moderate edi-ting (~25%) in DLB-1, while SPION-based magnetofection resulted in efficient uptake but no detectable editing, highlighting post-entry barriers. Confocal imaging and fluore-scence correlation spectroscopy revealed that nuclear localization and Cas9 aggregation are critical determinants of editing success. Our findings demonstrate that CRISPR/Cas9 delivery efficiency is cell line-dependent and governed by intracellular trafficking and genomic integrity. These insights provide a practical framework for optimizing gene editing in marine teleosts, advancing both basic research and selective breeding in aquaculture.

SUMMARY Neural crest induction begins early during neural plate formation, requiring precise transcriptional control to activate lineage-specific enhancers. Here, we demonstrate that SALL4, a transcription factor strongly expressed in cranial neural crest cells (CNCCs) and associated with syndromes featuring craniofacial anomalies, plays a critical role in this early regulatory process. Using a SALL4 -haploinsufficient human iPSC model that recapitulates clinical haploinsufficiency, we show that the SALL4A isoform directly interacts with the BAF complex subunit DPF2 through its zinc-finger-3 domain. This interaction enables SALL4-mediated recruitment of BAF to CNCC-specific enhancers during early neural plate stages. Without functional SALL4, BAF fails to load at chromatin, leaving CNCC enhancers inaccessible despite normal neuroectodermal progression. Consequently, cells cannot undergo proper CNCC induction and specification due to persistent enhancer repression. Our findings reveal SALL4 as an essential regulator of BAF-dependent enhancer activation during early neural crest development, providing molecular insights into SALL4-associated craniofacial anomalies.

Abstract Genome editing using CRISPR/Cas9 allows precise modifications within plants genomes, however, it also poses biosafety and biosecurity concerns, particularly regarding potential off-target mutations. Therefore, establishing robust detection methods is essential. A loop-mediated isothermal amplification (LAMP) assay coupled with Thioflavin T (ThT) fluorescence detection was developed for the sensitive and specific identification of the Cas9 coding sequence in genome-edited plants. Following optimization of reaction conditions—including ThT concentration, Mg²⁺ levels, and incubation time—the assay achieved a detection limit of approximately 4.34 copies/µL based on fluorescence analysis. Two G-rich sequences predicted from the LAMP amplicons were evaluated individually, revealing that one produced significantly higher fluorescence, suggesting a potential G-quadruplex (G4)-like conformation enhancing ThT signal. The method avoids colorimetric subjectivity and does not require real-time monitoring or complex instrumentation. Amplification products were confirmed via agarose gel electrophoresis, and fluorescence measurements were consistent across replicates. This study provided a isothermal assay to utilize ThT for Cas9 detection and offers a cost-effective, reliable platform for screening genome-edited organisms, particularly in settings with limited resources. The system shows strong potential for future application in biosafety and traceability.

Abstract CRISPR-Cas nucleases are transforming genome editing, RNA editing, and diagnostics but have been limited to RNA-guided systems. We present ΨDNA, a DNA-based guide for Cas12 enzymes, engineered for specific and efficient RNA targeting. ΨDNA mimics a crRNA but with a reverse orientation, enabling stable Cas12-RNA assembly and activating trans-cleavage without RNA components. ΨDNAs are effective in sensing short and long RNAs and demonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discovered that ΨDNAs can guide certain Cas12 enzymes for RNA targeting in cells, enhancing mRNA degradation via ribosome stalling and enabling multiplex knockdown of multiple RNA transcripts. This study establishes ΨDNA as a robust alternative to RNA guides, augmenting CRISPR-Cas12’s potential for diagnostic applications and for targeted RNA modulation in cellular environments.

Abstract CRISPR/Cas effectors rely on RNA guides to recognize target nucleic acids. In Cas9 and Cas12a systems, protospacer adjacent motif (PAM) on target DNA engage conserved residues in the Cas protein, thereby accelerating target binding and catalytic activation. Here, we overturn this paradigm by reprograming the canonical RNA-guided DNA-targeting machinery into a DNA-guided RNA-targeting platform. We engineered synthetic CRISPR DNA (crDNA) that emulates the PAM-duplex architecture to form stable deoxyribonucleoprotein complexes with Cas9 or Cas12a, redirecting their specificity towards RNA substrates. Coupling DNA-guided Cas12a with isothermal amplification enables attomolar detection of nucleic acid samples, while DNA-guided Cas9 achieves over 70% knock-down of EGFP expression in HEK293T cells. This DNA-guided paradigm reshapes our understanding of CRISPR targeting and paves the way for new diagnostic and therapeutic applications.

Vascular malformations are congenital lesions caused by somatic and germline mutations that disrupt developmental signaling pathways. Capillary malformations (CMs) typically present as port-wine stains in the skin and can also affect ocular and cerebral tissues in Sturge Weber Syndrome (SWS), leading to aesthetic, ophthalmic, and neurological complications. CMs are caused by a somatic mutation in the GNAQ gene in endothelial cells, leading to a p.R183Q substitution in the Gαq protein. The underlying mechanisms of Gαq-R183Q-driven CMs formation remain unclear. To address this, we generated CRISPR/Cas9-engineered human dermal microvascular endothelial cells lacking endogenous Gαq, whilst expressing the Gαq-R183Q mutant instead. The Gαq-R183Q mutation strongly impaired endothelial cell migration and angiogenic sprouting capacity compared to wild-type controls. Next, using SILAC-based quantitative proteomics, we investigated the Gαq-R183Q-induced changes in the endothelial phosphoproteome. These analyses revealed prominent activation of the calcineurin-NFAT signaling pathway in Gαq-R183Q-expressing endothelial cells, leading to dephosphorylation of NFAT1 and NFAT2 and the selective expression of their transcriptional target DSCR1.4. Immunofluorescence of patient-derived skin biopsies confirmed deregulation of NFAT1/2 and the expression of DSRC1 in endothelial cells, validating their potential importance in CMs. We further demonstrate that pharmacological inhibition of calcineurin with tacrolimus (FK506) could partially restore NFAT signaling in Gαq-R183Q endothelial cells. Intriguingly, the genetic depletion of the NFAT target DSCR1 in Gαq-R183Q cells fully restored calcineurin/NFAT signaling to normal levels, enabling proper endothelial migration and sprouting. In summary, we uncovered a calcineurin-NFAT-DSCR1.4 signal transduction axis that is driven by Gαq-R183Q and established its importance for endothelial angiogenic properties. These findings highlight the calcineurin/NFAT signaling axis as a promising therapeutic target to restore endothelial function in CMs.

Flaviviruses such as dengue and Zika viruses extensively remodel host cell membranes to create specialised replication organelles, but the molecular mechanisms governing lipid metabolism during infection remain poorly understood. Through systematic screens of fatty acyl transferase enzymes (MBOAT and zDHHC families) and complementary approaches including CRISPR/Cas9 gene deletions, pharmacological inhibition, proteomics, and photo-crosslinkable cholesterol analogues, we identified Sterol O-acyltransferases 1 and 2 (SOAT1/SOAT2) as critical host dependency factors for flavivirus infection. SOAT1/2 activities were upregulated early during infection, coinciding with increased LD formation, which underwent transition to liquid crystalline phases. Genetic deletion or pharmacological inhibition of either enzyme resulted in a dramatic ∼100-fold reduction in viral production. Mechanistically, SOAT1/2 generate cholesteryl ester-enriched lipid droplets with fundamentally altered proteomes, enriched in fatty acid remodelling enzymes, Rab-GTPases, lipid transport proteins and sphingomyelinases. Photo-crosslinking experiments demonstrated direct interactions between LD-derived cholesterol and viral prM, capsid and NS1. SOAT1/2 deficiency resulted in defective, viral RNA-free replication organelles and complete absence of immature virions. Supporting the clinical relevance of viral lipid exploitation, analysis of dengue patients from a Sri Lankan cohort revealed that central obesity significantly increased the risk of severe dengue haemorrhagic fever, compared to lean patients. This study establishes SOAT1/2 as essential host factors that enable flavivirus morphogenesis through specialised cholesteryl ester-enriched LDs, revealing a previously unrecognised virus-host interaction mechanism and identifying host lipid metabolism as a promising therapeutic target for combating flavivirus infections.

ABSTRACT Nephronophthisis (NPH) is a heterogeneous, autosomal recessive ciliopathy and an important cause of end-stage renal disease (ESRD) in children and young adults. Since its classification as ciliopathy in 2003, NPH disease causal attribution had been focused primarily on ciliary dysfunction. The finding that ciliopathy players are involved in the DNA damage response (DDR) signaling resulted in a paradigm shift in thinking on NPH disease aetiology. Mutations in NPHP1 are the leading cause of NPH, but the underlying mechanisms that lead to the disease phenotype remain poorly understood. Here, nephrocystin-1 depleted kidney organoids were generated and characterized to address this knowledge gap. We used CRISPR/Cas9 to generate NPHP1 control ( NPHP1 WT ) and two mutant ( NPHP1 ko1 and NPHP1 ko2. ) cell lines from healthy human induced pluripotent stem cells (iPSC), differentiated into kidney organoids in an air-liquid interface following an optimized protocol. Upon loss of nephrocystin-1, kidney organoids showed impaired nephron structures and loss of glomerular mesangial and distal tubular cells. Furthermore, NPHP1 depleted organoids exhibited a persistent inability to repair DNA lesions and showed increased senescence and fibrosis characteristics. Dynamic subcellular localization of nephrocystin-1 in NPHP1 WT , particularly its translocation to nuclei 15 min post-UVC light exposure, suggested its direct involvement in the DDR. In conclusion, a novel NPHP1 -depleted kidney organoid model was established, providing a platform to comprehensively study DNA damage, senescence and fibrosis simultaneously upon nephrocystin-1 loss. This advanced model aids in the understanding of the pathophysiology of NPH and paves the way towards identifying novel druggable targets.

ABSTRACT Botrytis cinerea is recognized as one of the most harmful fungal diseases affecting grapevine ( Vitis vinifera ), directly impacting grape yield and wine quality. Identifying new genes involved in the interaction between V. vinifera and B. cinerea appears to be the most promising strategy for enhancing grapevine resistance to this pathogen in future breeding programs. During pathogen infection, plasma membrane-localized pattern recognition receptors (PRRs) are involved in the perception of conserved microbe-associated molecular patterns (MAMPs). Among PRRs, members of the LysM receptor-like kinase family are well known to mediate recognition of fungal MAMPs to induce the plant immune signaling pathway. Interestingly, a novel member of this receptor family, named VvLYK6, was identified in grapevine as the most upregulated during a Botrytis cinerea infection. Aim: ing to understand the role of VvLYK6 in plant immunity, we carried out an overexpression study in Arabidopsis thaliana and in grapevine cell suspension. The overexpression of VvLYK6 resulted in a reduction of chitin oligomer-induced MAPKs activation, expression of defense-related genes, a reduced callose deposition and an increased plant susceptibility to three fungal pathogens. At the opposite, the CRISPR-Cas9-mediated vvlyk6 knock-out lines generated in V. vinifera induce the phytoalexin-related stilbene pathway at the basal level. Based on our findings, we concluded that VvLYK6 negatively regulates the chitin-triggered immune responses in V. vinifera , suggesting its potential involvement as a susceptibility gene during fungal infections.

Abstract Molnupiravir, a nucleoside analog effective against RNA viruses such as Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2), exerts antiviral effects primarily through lethal mutagenesis by incorporation into viral RNA. However, its ambiguous base-pairing properties raise concerns about potential mutagenesis via conversion to deoxycytidine diphosphate (dCDP) by cellular ribonucleotide reductase (RNR) and subsequent incorporation into DNA. This study evaluates whether herpes simplex virus type 1 (HSV-1), in comparison to HSV-1 lacking RNR (HSV-1 ΔRR), can serve as a model to investigate molnupiravir’s mutagenic effects on DNA viruses. Using CRISPR-Cas9, the ICP6 RNRdomain has already been deleted to generate HSV-1 ΔRR. Human primary fibroblasts with low endogenous deoxyribonucleotide pools were infected at low multiplicity and treated with varying concentrations of molnupiravir. Viral titers were measured by TCID50 assays, and viral DNA from treated cultures was sequenced, targeting nonessential genes to detect mutations. Molnupiravir significantly reduced viral titers of wild-type HSV-1 in both fibroblasts and Vero cells, while HSV-1 ΔRR exhibited reduced sensitivity, with significant inhibition only at higher drug concentrations in fibroblasts. Importantly, no mutations were detected in viral DNA from either strain at any molnupiravir concentration. These findings indicate that molnupiravir’s mutagenic potential is limited in DNA viruses like HSV-1, likely due to restricted incorporation into DNA. This highlights the need for further research to optimize molnupiravir’s antiviral use beyond RNA viruses.

Rationale Chimeric antigen receptor (CAR) T cell therapies have shown remarkable success in treating hematological cancers and are increasingly demonstrating potential for solid tumors. CRISPR-based genome editing offers a promising approach to enhance the potency and safety of CAR-T cells. However, several challenges persist, including inefficient tumor homing and treatment-related toxicities in normal tissues, which continue to hinder widespread adoption. Advanced imaging technologies, including bioluminescence imaging (BLI) and positron emission tomography (PET), provide real-time insights into CAR-T cell distribution and activity in vivo, both in preclinical models and in patients. Here, we developed Trackable Reporter Adaptable CRISPR-Edited CAR (tRACE-CAR) T cells, a modular system for site-specific integration of CARs and imaging reporters. Methods The luciferase reporter AkaLuciferase (AkaLuc) or the human sodium iodide symporter (NIS) were cloned downstream of the CAR in adeno-associated virus (AAV) donors for BLI or PET tracking, respectively. CARs with imaging reporters were knocked into the TRAC locus of primary human T cells via CRISPR editing and AAV transduction. Editing efficiency was evaluated by flow cytometry and junction PCR. In vitro cytotoxicity was assessed by BLI using firefly luciferase (Fluc)-expressing cancer cells co-cultured with CAR-T cells at varying effector-to-target ratios. In vivo, BLI and PET imaging assessed CAR-AkaLuc and CAR-NIS T cell expansion and trafficking in Nod-SCID-gamma mice bearing xenograft tumors. Results T cell receptor (TCR) knockout efficiency exceeded 85%, with CAR expression observed in 70–80% of cells, depending on the reporter used. Reporter-engineered CAR-T cells retained functionality in vitro and exhibited significant cytotoxicity against target cancer cells, outperforming naïve T cells. In vivo, AkaLuc BLI and 18 F-tetrafluoroborate PET enabled non-invasive tracking of viable CAR-T cells. Notably, the route of administration (intravenous, peritumoral, or intraperitoneal) significantly influenced the distribution of CAR-T cells and their therapeutic effectiveness. Conclusion tRACE-CAR enabled precise optical and PET tracking of CAR-T cells in models of B cell leukemia and ovarian cancer, allowing dynamic, non-invasive monitoring of cell distribution in both tumors and off-target tissues. This imaging platform could lead to more personalized, effective CRISPR-edited CAR cell therapies.

Functional regeneration of volumetric muscle loss (VML) remains a significant challenge in the field of regenerative medicine. We have developed a novel biomimetic scaffold (0.4 mm thick hernia patch) derived from soluble collagen. This biomimetic scaffold exhibits exceptional strength, low immunogenicity, and structurally mimics the natural muscle fiber arrangement. The scaffold is utilized to repair a VML rabbit model (30 × 30 mm defect in abdominal wall) using nylon sutures for connection. It was observed that the VML site was gradually covered by regenerated tissue, which consisted of 2-3 mm thick functional muscle. By 32 weeks post-surgery, the newly formed muscle tissue had covered the majority of the VML area, as evidenced by morphological observations and histological evaluations. Approximately 24 weeks after surgery, the scaffold underwent complete degradation. This degradation exhibited a strong correlation with muscle regeneration. Furthermore, it was observed that the nylon sutures gradually migrated towards the VML center as muscle regeneration progressed, and nylon sutures exhibited an adverse impact on muscle regeneration.. The study did not employ exogenous cells or growth factors. However, the collagen scaffold effectively stimulated endogenous muscle regeneration. In contrast to the previous limited partial recovery or fibrotic scar healing, the collagen scaffold successfully induced genuine structural and functional muscle regeneration. The 0.4 mm thick scaffold (hernia patch) exhibits a different structure from the 2-3 mm thick natural abdominal muscle wall. This suggests that a simple scaffold can regenerate functional tissue with a complex structure. This research represents the first successful functional regeneration of VML in clinically relevant animal models through the utilization of artificial materials. This technology possesses transformative potential in the treatment of muscle defects arising from trauma, tumor resection, hernia, or congenital anomalies. Furthermore, it holds substantial medical potentials in addressing neuromuscular diseases, such as Duchenne muscular dystrophy (DMD), amyotrophic lateral sclerosis (ALS), and spinal muscular atrophy (SMA), by collaborating with advanced technologies including CRISPR-Cas9, adeno-associated virus (AAV) gene therapy, and stem cell technology.