CRISPR-associated transposons (CAST) consist of an integration between certain class 1 or class 2 CRISPR-Cas systems and Tn7-like transposons. Class 2 type V-K CAST systems are restricted to cyanobacteria. Here, we identified a unique subgroup of type V-K systems through phylogenetic analysis, classified as V-K_V2. Subgroup V-K_V2 CAST systems are characterized by an alternative tracrRNA, the exclusive use of Arc_2-type transcriptional regulators, and distinct differences in TnsB and TnsC proteins. Although the occurrence of V-K_V2 CAST systems is restricted to Nostocales cyanobacteria, it shows signs of horizontal gene transfer, indicating its capability for genetic mobility. The predicted V-K_V2 tracrRNA secondary structure has been integrated into an updated version of the CRISPRtracrRNA program available on GitHub under https://github.com/BackofenLab/CRISPRtracrRNA/releases/tag/2.0 .

Abstract The accumulation of misfolded protein species underlies a broad range of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Due to their dynamic nature, these misfolded proteins have proven challenging to target therapeutically. Here, we specifically target misfolded disease variants of the ALS-associated protein; SOD1, using a biological proteolysis targeting chimera (BioPROTAC) composed of a SOD1-specific intrabody and an E3 ubiquitin ligase. Screening of intrabodies and E3 ligases for optimal BioPROTAC construction revealed a candidate capable of degrading multiple disease variants of SOD1, preventing their aggregation in cultured cells. Using CRISPR/Cas9 technology to develop a BioPROTAC transgenic mouse line, we demonstrate that the presence of the BioPROTAC delays disease progression in the SOD1G93A mouse model of ALS. Delayed disease progression was associated with protection of motor neurons, reduced accumulation of insoluble SOD1 and protection of neuromuscular junctions. These findings provide proof-of-concept evidence and a platform for developing BioPROTACs as a therapeutic strategy for the targeted degradation of neurotoxic misfolded species in the context of neurodegenerative diseases.

Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic var-iants in ALPL, coding for tissue non-specific alkaline phosphatase. HPP patients suffer from impaired bone mineralization and in severe cases from vitamin B6-responsive sei-zures. To study HPP we generated alpl-/- zebrafish line using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf) no alpl mRNA and 89% lower total alkaline phosphatase activity was detected in alpl-/- compared to alpl+/+ embryos. The survival of alpl-/- zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in alpl-/- embryos. B6 vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in alpl-/- embryos. Accumulation of d3-pyridoxal 5’-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in alpl-/- embryos incu-bated with d3-PLP confirmed Alpl involvement in vitamin B6 metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in alpl-/- embryos. Metabolic profiling of alpl-/- larvae using direct-infusion high-resolution mass spectrome-try showed abnormalities in polyamine and neurotransmitter metabolism, suggesting dysfunction of vitamin B6-dependent enzymes. Accumulation of N-methylethanolaminium phosphate indicated abnormalities in phosphoethanolamine metabolism. Taken together, we generated first zebrafish model of HPP that shows multi-ple features of human disease and is suitable to study pathophysiology of HPP and to test novel treatments.

Intratumor transcriptional heterogeneity (ITTH) presents a major challenge in cancer treatment, particularly due to limited understanding of the diverse malignant cell populations and their relationship to therapy resistance. While single-cell sequencing has provided valuable insights into tumor composition, its high cost and technical complexity limits its use for large-scale tumor screening. In contrast, several databases collecting bulk RNA sequencing (RNA-seq) data from multiple samples across various cancer types are available and could be used to profile ITTH. Several deconvolution approaches have been developed to infer cellular composition from such data. However, most of these methods rely on predefined markers or single cell reference datasets, limiting the performance of such methods by the quality of used reference data. Although unsupervised approaches do not face such limitations, existing methods have not been specifically adapted to characterize malignant cell states, and focus instead on general cell types. To address these gaps, we introduce CDState, an unsupervised method for inferring malignant cell subpopulations from bulk RNA-seq data. CDState utilizes a Nonnegative Matrix Factorization model improved with sum-to-one constraints and a cosine similarity-based optimization to deconvolve bulk gene expression into distinct cell state-specific profiles, and estimate the abundance of each state across tumor samples. We validate CDState using bulkified single-cell RNA-seq data from five cancer types, showing that it outperforms existing unsupervised deconvolution methods in both cell state proportions and gene expression estimation. Applying CDState to 33 cancer types from TCGA, we identified recurrent malignant cell programs, with epithelial-mesenchymal transition as the main driver of tumor transcriptional heterogeneity. We further link the identified malignant states to patient clinical features, revealing states associated with worse patient prognosis. We show that ITTH is linked with patient survival and clinical features, as well as is associated with varied responses to therapy. Finally, we identified potential genetic drivers of ITTH and malignant cell states, including TP53 , KRAS , and PIK3CA , known oncogenic genes.

Cis-regulatory elements (CREs) control how genes respond to external signals, but the principles governing their structure and function remain poorly understood. While differential transcription factor binding is known to regulate gene expression, how CREs integrate the amount and combination of inputs to secure precise spatiotemporal profiles of gene expression remains unclear. Here, we developed a high-throughput combinatorial screening strategy, that we term NeMECiS, to investigate signal- dependent synthetic CREs (synCREs) in differentiating mammalian stem cells. By concatenating fragments of functional CREs from genes that respond to Sonic Hedgehog in the developing vertebrate neural tube, we found that CRE activity follows hierarchical design rules. While individual 200-base-pair fragments showed minimal activity, their combinations generated thousands of functional signal-responsive synCREs, many exceeding the activity of natural sequences. Statistical modelling revealed CRE function can be decomposed into specific quantitative contributions in which sequence fragments combine through a multiplicative rule, tuned by their relative positioning and spacing. These findings provide a predictive framework for CRE redesign, which we used to engineer synthetic CREs that alter the pattern of motor neuron differentiation in neural tissue. These findings establish quantitative principles for engineering synthetic regulatory elements with programmable signal responses to rewire genetic circuits and control stem cell differentiation, providing a basis for understanding developmental gene regulation and designing therapeutic gene expression systems.

We established a novel knock-in technique, New and Easy Xenopus Targeted integration ( NEXTi ), to recapitulate endogenous gene expression by reporter expression. NEXTi is a CRISPR-Cas9-based method to integrate a donor DNA containing a reporter gene ( egfp ) into target 5′ untranslated region (UTR) of Xenopus laevis genome. It enables us to track eGFP expression under regulation of endogenous promoter/enhancer activities. We obtained about 2% to 13% of knock-in embryos showing eGFP signal in a tissue-specific manner, targeting krt.12.2.L , myod1.S , sox2.L and bcan.S loci, as previously reported. In addition, F1 embryos which show stable eGFPs signal were obtained by outcrossing multiple founders with wild type animals. Integrations of donor DNAs into target 5′ UTRs were confirmed by PCR amplification and sequencing. Here, we describe the step-by-step protocol for preparation of donor DNA and single guide RNA, microinjection and genotyping of F1 animals for the NEXTi procedure.

Hypoxia, a hallmark of solid tumors, promotes the malignant progression and is challenging to target. Metabolic reprogramming and the resulting metabolic vulnerabilities provide a promising strategy to target tumor hypoxia. Here we systematically compared the metabolic network differences between hypoxic and non-hypoxic cells, and developed a deep learning model, “DepFormer”, to predict the dependent metabolic genes in hypoxic tumor cells. The performance of DepFormer was validated using CRISPR screening dataset. Oxidative phosphorylation was identified as the most significantly hypoxia-dependent metabolic pathway, and FLAD1 was predicted to be one of the key hypoxia-dependent metabolic genes. FLAD1 locus is amplified, and FLAD1 expression is upregulated in various tumor types, especially in hypoxic tumors. FLAD1 depletion compromises tumor’s adaptation to hypoxia by disrupting mitochondrial complex II activity, leading to an imbalance between succinate and fumarate, and consequent failure to adapt to hypoxia. Subsequently, we identified a drug-like inhibitor of FLAD1, which selectively inhibits the growth of tumor cells under hypoxia. Our findings reveal FLAD1 as an innovative therapeutic target for hypoxic tumors.

Soybean transformation remains challenging and has not kept pace with the rapid advancement of genetic engineering technologies due to low efficiency, lengthy timelines, and genotype dependency. Here, we developed a streamlined transformation method by leveraging developmental regulators (DRs) to promote de novo shoot regeneration directly from growing soybean plants. By evaluating multiple DR combinations, our results showed that co-expression of WUSCHEL2 ( WUS2 ) and isopentenyltransferase ( IPT ) achieved higher transformation efficiencies (15.2% to 22.3%) in Williams 82 and Bert varieties than individual DRs without requiring exogenous hormones or selection agents. Moreover, this method produces heritable transgenic events within 9-11 weeks and successfully delivers CRISPR-Cas9 components, generating heritable mutations with 20% efficiency. The temporal transcriptomic and gene regulatory network analyses revealed that WUS2 / IPT synergistically modulates stress responses and activates developmental pathways, orchestrating a transition from initial stress adaptation to regenerative programming. Together, our findings demonstrate that this DR-enabled approach significantly enhances soybean transformation efficiency, reduces tissue culture requirements, and offers a promising genome editing platform for soybean improvement.

Background Calcific aortic valve stenosis (AS) affects 3% of older adults and lacks medical treatment. The deacetylase Sirtuin 1 (SIRT1) could be involved in many pathways linked to AS progression. Sodium-glucose co-transporter 2 inhibitors (SGLT2i), glucose-lowering agents, have been shown to reduce cardiovascular events (likely via SIRT1), but their possible benefits in AS are unknown. Our study aims to uncover the role of SIRT1 in AS progression and assess the benefit of SGLT2i to slow down the aortic valve fibro-calcification processes. Methods RNA-seq data of human aortic valve specimens were collected from ARChS4 database. SIRT1 knockdown (SIRT1 KD) and overexpressing (SIRT1 Over) valve interstitial cells (VIC) were generated by CRISPR/Cas9. Real-time PCR, immunofluorescence, and calcification assays were used to characterized mutant VICs. Conditioned medium experiments were implemented to evaluate SGLT2i effect on cellular cross-talk and calcification. Diabetic patients’ data from the Lombardy regional healthcare database, treated with sulphonylureas (SU; no effect on SIRT1) and SGLT2i (acting on SIRT1), were selected and matched 1:1 by age, sex, and multisource comorbidity score. Cumulative incidence of hospitalization for non-rheumatic aortic valve disease was assessed by Kaplan-Meier and multivariable Cox proportional hazards models were used to estimate hazard ratios. Results RNA-seq showed that SIRT1 could be a master regulator of multiple AS-related pathways. Functional studies on mutant VICs revealed that SIRT1 directly regulates antioxidant processes, extracellular-matrix remodeling and calcification by modulating key transcription factors. Moreover, calcification assays further support this role, revealing an increased calcification in SIRT1 KD VICs and a concomitant decrease in VIC SIRT1 Over when compared to wild type. Then, exploring SGLT2i impact on calcification, we showed that VICs cultured in SGLT2i-treated-endothelial medium exhibited reduced calcification associated with endothelial-increased nitric oxide levels, while SIRT1 inhibition enhanced VIC calcification. The real-world data analysis revealed that SGLT2i-treated group had a lower incidence of hospitalized patients for non-rheumatic aortic valve disease compared to SU-treated group. Conclusions Our data identify SIRT1 as a key regulator of fibro-calcific processes in AS and suggest that SGLT2i may slow the aortic valve degeneration through SIRT1 modulation. These findings highlight SGLT2i as a promising therapeutic option for AS prevention and care. Clinical Perspectives What is new? Sirtuin 1 (SIRT1) downregulation is linked to the progression of aortic stenosis (AS) pathological processes and plays a crucial role in mitigating oxidative stress, fibrosis, and calcification. Sodium-glucose co-transporter 2 inhibitor (SGLT2i) treatment of valve endothelial cells results in the secretion of protective factors that reduce valve interstitial cell calcification, highlighting the valuable role of endothelial health in preventing valve degeneration. Real-world data indicate that the use of SGLT2i is associated with a lower incidence of hospitalization rate for aortic valve disease as compared to sulfonylureas, suggesting a protective effect against AS in diabetic patients. What are the clinical implications? This study highlights the potential of restoring SIRT1 activity as a therapeutic strategy to mitigate pro-calcific and pro-fibrotic processes in AS. SGLT2i may offer a breakthrough therapeutic option for AS, a condition currently lacking effective pharmacological treatments, providing new hope for slowing disease progression and improving clinical outcomes.

1 Characterization of essential genes across the genome is fundamental to understanding cellular functions at a molecular level. While significant progress has been made in characterizing essential genes in human and mouse models, relatively little is known about essential genes in the porcine genome. Pigs are an important production species and are now emerging as valuable models for studying human diseases due to their physiological similarities to humans. To map essential genes across the porcine genome, we have developed a novel porcine genome-wide CRISPR knockout screening library (pGeCKO) and applied it to two porcine cell lines, PK15 and IPEC-J2. We identified 2,245 essential genes in PK15 cells and 919 essential genes in IPEC-J2 cells, with 683 of these shared between both cell lines. Functional analyses revealed that most essential genes are involved in core cellular processes such as cell cycle regulation, DNA replication, transcription, and translation. Comparative analysis with human essential genes from the DepMap project revealed that over half of the genes are shared with humans and the rest are porcine-specific. These porcine-specific essential genes included genes in core functional pathways related to protein and RNA processing as well as many related to N-glycan biosynthesis, signal transduction, and several long-noncoding RNAs. This work provides a new resource for leveraging porcine models in disease research, enhancing our understanding of porcine genetics and its implications for human health.

PML nuclear bodies (PML-NBs) are dynamic subnuclear structures important for chromatin dynamics and anti-viral defense. In this study we investigate the role of Sp100 isoforms in promoting localization of the H3.3 histone chaperone HIRA to PML-NBs in human keratinocytes. Sp100 knockout (KO) cell lines were generated using CRISPR-Cas9 technology and shown to display normal keratinocyte differentiation and PML-NB formation. However, HIRA and its associated complex members (UBN1 and ASF1a) failed to localize to PML-NBs in the absence of Sp100, even after interferon stimulation. Exogenous expression of the four main isoforms of Sp100 showed that the Sp100A isoform is the primary driver of HIRA localization to PML-NBs, with the SUMO interacting motif (SIM) playing an important role. These findings highlight the functional diversity of the Sp100 isoforms in modulating chromatin dynamics at PML-NBs.

ABSTRACT Serine incorporator 5 (SERINC5) is a host restriction factor that targets certain enveloped viruses, including human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). It integrates into the viral envelope from the cell surface, inhibiting viral entry. SERINC5 is transported to the cell surface via polyubiquitination, while a single K130R mutation retains it in the cytoplasm. Both HIV-1 Nef and MLV glycoGag proteins antagonize SERINC5 by reducing its expression in producer cells. Here, we report that MLV glycoGag employs selective autophagy to downregulate SERINC5, demonstrating a more potent mechanism for decreasing its cell surface expression. Although glycoGag is a type II integral membrane protein, it primarily localizes to the cytoplasm and undergoes rapid proteasomal degradation. Employing the K130R mutant, we show that Nef, primarily associated with the plasma membrane, downregulates SERINC5 only after it has trafficked to the cell surface, whereas glycoGag can reduce its expression before reaching the plasma membrane while still in the cytoplasm. Nonetheless, an interaction with SERINC5 stabilizes and recruits glycoGag to the plasma membrane, enabling it to downregulate SERINC5 from the cell surface. Through affinity-purified mass spectrometry analysis combined with CRISPR/Cas9 knockouts, we find that glycoGag’s activity depends on reticulophagy regulator 1 (RETREG1), an ER-phagy receptor. Further knockout experiments of critical autophagy genes demonstrate that glycoGag downregulates cytoplasmic SERINC5 via micro-ER-phagy. These findings provide crucial new insights into the ongoing arms race between retroviruses and SERINC5 during infection. AUTHOR SUMMARY HIV-1 Nef and MLV glycoGag are unrelated viral proteins, yet both counteract the same host restriction factor, SERINC5, to facilitate productive infection. In this study, we report a novel pathway through which glycoGag downregulates SERINC5. We demonstrate that while Nef downregulates SERINC5 only after it has trafficked to the cell surface, glycoGag can directly downregulate SERINC5 in the cytoplasm before it reaches the plasma membrane. Furthermore, we show that this pathway is mediated by the ER-phagy receptor RETREG1, which targets SERINC5 for degradation via micro-ER-phagy. This mechanism provides a more effective means of blocking SERINC5 antiviral activity. These findings reveal that retroviruses have evolved different strategies to antagonize SERINC5, highlighting the critical role of SERINC5 in restricting retroviral infections.

ABSTRACT Producing the gene-knockout mutant is a critical strategy in reverse genetics for gene functional analyses. Plant science has applied various mutagenesis, including chemical mutagen, T-DNA insertion, and genome editing. The intact gene expression is completely disrupted, while the mutant retains the intact sequence region. Lately, effective whole open reading frame (ORF) deletion through the CRISPR/Cas9 system using multiplex guide RNAs was reported in Arabidopsis, targeting a gene expressed in somatic cells. Here we applied the scheme targeting the reproductive genes GENERATIVE CELL SPECIFIC 1 ( GCS1 ), GAMETE EXPRESSED 2 ( GEX2 ), DUF679 DOMAIN MEMBRANE PROTEIN 8 ( DMP8 ), and DMP9 , which are fertilization regulators. Homozygous gene deletion lines were successfully obtained in the T1 generation, with acquisition rates ranging from 8.3% to 30.0%. The rates approximately correlated with the scores predicted by the DeepSpCas9. The analysis of the GCS1 deletion line ( Δgcs1 ) suggested that avoiding the first bolt removal is important for consistent phenotype of the developed inflorescences. Compared to the previously reported mutants, the GEX2 deletion line ( Δgex2 ) showed a difference in seed development phenotype, indicating that the remaining intact gene region in the mutant could unexpectedly influence the function. Simultaneous deletions of DMP8 and DMP9 , which are in distinct chromosomes, were also succeeded in the T1 generation. The obtained results showed that all deletions were inheritable. The scheme challenged here demonstrated the effective production of homozygous mutants for the genes, even if the reproductive lethal or recessive.

ABSTRACT The phenotype of a mutation often differs across genetically distinct individuals. In the most extreme case, a gene can be essential for viability in one genetic background, but dispensable in another. Although genetic context-dependency of mutant phenotypes is frequently observed, the underlying causes often remain elusive. Here, we investigated the genetic changes responsible for differences in gene essentiality across 18 genetically diverse natural yeast strains. First, we identified 39 genes that were essential in the laboratory reference strain but not required for viability in at least one other genetic background, suggesting that the natural strain contained suppressor variants that could bypass the need for the essential gene. We then mapped and validated the causal bypass suppressor variants using bulk segregant analysis and allele replacements. Bypass suppression was generally driven by a single modifier gene that tended to differ between genetic backgrounds. The suppressors often indirectly counteracted the effect of deleting the essential gene, for instance by changing the transcriptome of a cell. Context-dependent essential genes and their bypass suppressors were frequently co-mutated across 1,011 yeast isolates and identified naturally occurring evolutionary trajectories. Overall, our results highlight the relatively high frequency of bypass suppression in natural populations, as well as the underlying variants and mechanisms. A thorough understanding of the causes of genetic background effects is crucial for the interpretation of genotype-to-phenotype relationships, including those associated with human disease.

Understanding how genetic variants drive phenotypic differences is a major challenge in molecular biology. Single nucleotide polymorphisms form the vast majority of genetic variation and play critical roles in complex, polygenic phenotypes, yet their functional impact is poorly understood from traditional gene-level analyses. In-depth knowledge about the impact of single nucleotide polymorphisms has broad applications in health and disease, population genomic and evolution studies. The wealth of genomic data and available functional genetic tools make Drosophila melanogaster an ideal model species for studies at single nucleotide resolution. However, to leverage these resources for genotype-phenotype research and potentially combine it with the power of functional genetics, it is essential to develop techniques to predict functional impact and causality of single nucleotide variants. Here, we present FlyCADD, a functional impact prediction tool for single nucleotide variants in D. melanogaster. FlyCADD, based on the Combined Annotation-Dependent Depletion (CADD) framework, integrates over 650 genomic features - including conservation scores, GC content, and DNA secondary structure - into a single metric reflecting a variants predicted impact on evolutionary fitness. FlyCADD provides impact prediction scores for any single nucleotide variant on the D. melanogaster genome. We demonstrate the power of FlyCADD for typical applications, such as the ranking of phenotype-associated variants to prioritize variants for follow-up studies, evaluation of naturally occurring polymorphisms, and refining of CRISPR-Cas9 experimental design. FlyCADD provides a powerful framework for interpreting the functional impact of any single nucleotide variant in D. melanogaster, thereby improving our understanding of genotype-phenotype connections.

A temperate N-15-like phage and an extensively drug-resistant (XDR) Klebsiella pneumoniae strain were studied in this research. The former was found in hospital wastewater, while the latter was retrieved from the sputum of an intensive care unit patient. The bacteria showed strong resistance to several antibiotics, including penicillin (≥16 μg/mL), ceftriaxone (≥32 μg/mL), and meropenem (≥8 μg/mL), which was caused by SHV-11 beta-lactamase, NDM-1 carbapenemase, and porin mutations (OmpK37, MdtQ). Yersiniabactin, enterobactin, and E. coli common pilus (ECP) genes were also present in the genome; these genes are essential for the acquisition of iron, adhesion, and immune evasion, among other virulence factors. kappa testing categorized the strain as K64 and O2a types. The presence of colicin genes, IncHI1B_1_pNDM-MAR and IncFIB replicons, and other plasmids in this strain demonstrate its ability to spread antibiotic resistance and facilitate horizontal gene transfer. Adding to its genetic variety and adaptability, the genome included CRISPR-Cas systems and eleven prophage regions. The 172,025 bp linear genome and 46.3% GC content of the N-15-like phage showed strong genomic similarities to phages of the Sugarlandvirus genus, especially those that infect K. pneumoniae. There were structural proteins (11.8 percent of ORFs), DNA replication and repair enzymes (9.3 percent of ORFs), and a toxin-antitoxin system (0.4 percent of ORFs) encoded by the phage genome. A protelomerase and ParA/B partitioning proteins indicate that the phage is replicating and maintaining itself in a manner similar to the N15 phage, which is renowned for maintaining a linear plasmid prophage throughout lysogeny. Lysogeny and horizontal gene transfer are two mechanisms by which phages may influence bacterial evolution. Learning about the phage’s role in bacterial evolution, host-phage relationships, and horizontal gene transfer is a great benefit. Understanding the dynamics of antibiotic resistance and pathogen development requires knowledge of phages like this one, which are known for their temperate nature and their function in altering bacterial virulence and resistance profiles. The regulatory and structural proteins of the phage also provide a model for research into the biology of temperate phages and their effects on microbial communities. The importance of temperate phages in bacterial genomes and their function in the larger framework of microbial ecology and evolution is emphasized in this research. Author Summary Antibiotic-resistant bacteria represent a significant global health threat, and comprehending their interactions with bacteriophages is essential for formulating novel antimicrobial tactics and elucidating the molecular development of bacteria. This work examined an extensively drug-resistant (XDR) strain of Klebsiella pneumoniae isolated from an ICU patient and a temperate N-15-like phage identified in hospital effluent. The bacterial strain exhibited resistance to multiple antibiotics owing to an array of resistance genes, plasmids, porin mutations, and virulence characteristics that facilitated its survival and pathogenicity. The genomic investigation of the phage elucidated its structural organization, replication mechanisms, and possible contribution to bacterial development through lysogeny and horizontal gene transfer. Our findings underscore the intricate host-phage interactions that affect antibiotic resistance and pathogenicity in K. pneumoniae, offering significant insights into microbial evolution and the prospective relevance of phages in therapeutic approaches.

Abstract Various cellular stresses release immunogenic molecules and elicit immunosurveillance to detect and eliminate potential threats, thus safeguarding organismal health. Nucleolar stress is characterized by the disruption of nucleolar morphology and function, leading to impaired ribosome biogenesis and activation of stress response pathways. However, the regulatory mechanisms linking nucleolar stress to tumour-intrinsic immunogenicity and its therapeutic implications remain unclear. Utilizing CRISPR screens to identify cancer-specific immunogenic regulators, we identified nucleolar OTUD4 (an ovarian tumour domain-containing deubiquitinase) as a repressor of immunogenicity by preserving nucleolar homeostasis. OTUD4 depletion induced nucleolar stress, thereby enhancing immunogenicity and retarding tumor growth in preclinical models. Mechanistically, OTUD4 functioned as a phospho-activated K63 deubiquitinase, catalyzing the K63-linked deubiquitination of nucleophosmin 1 (NPM1) to stabilize its oligomerization. Consequently, OTUD4 deletion promoted NPM1 hyperubiquitination and depolymerization, thereby driving the release of NPM1-binding heterochromatin H3K9me3. The heterochromatin remodeling derepressed endogenous retroelements (ERV) that subsequently stimulated cytosolic DNA-sensing pathways to trigger the type I interferon response and upregulate the expression of antigen presentation genes. Concurrently, ERV-encoded retroviral antigens augmented tumour-intrinsic antigenicity. A pharmacological screen revealed that the casein kinase II inhibitor CX-4945 phenocopied OTUD4 ablation by dephosphorylating and inactivating OTUD4 activity. Clinically, OTUD4 amplification or a high OTUD4-nucleolus gene signature inversely correlated with patient survival and immunotherapy resistance. Our findings position nucleolar stress as a central driver of tumour-intrinsic immunogenicity, and propose a clinical rationale for leveraging nucleolar stress inducers to sensitize immunologically cold tumours to immunotherapy.

Antimicrobial resistance (AMR) threatens global health. In this manuscript, I review recent literature underscoring the promise of engineered bacteriophages and CRISPR-Cas systems as targeted strategies against resistant bacteria. These approaches offer alternatives to broad-spectrum antibiotics by precisely disrupting biofilms and inactivating resistance genes—whether applied independently or in tandem. I also underscore the essential role of public-private partnerships in advancing clinical applications and catalyzing the translation of innovative research into practice

CD19-CAR-T-cells emerge as a major therapeutic option for relapsed/refractory B-cell-derived malignancies, however approximately half of patients eventually relapse. To identify resistance-driving factors, we repeatedly exposed B-cell lymphoma/B-cell acute lymphoblastic leukemia to 4-1BB/CD28-based CD19-CAR-T-cells in vitro . Generated models revealed costimulatory domain-dependent differences in CD19 loss. While CD19-4-1BB-CAR-T-cells induced combination epitope/total CD19 protein loss, CD19-CD28-CAR-T-cells did not drive antigen-escape. Consistent with observations in patients relapsing after CD19-4-1BB-CAR-T-cells, we identified CD19 frameshift/missense mutations affecting residues critical for FMC63 epitope recognition. Mathematical simulations revealed that differences between CD19-4-1BB- and CD19-CD28-CAR-T-cells activity against low-antigen-expressing tumor contribute to heterogeneous therapeutic responses. By integrating in vitro and in silico data, we propose a biological scenario where CD19-4-1BB-CAR-T-cells fail to eliminate low-antigen tumor cells, fostering CAR-resistance. These findings offer mechanistic insight into the observed clinical differences between axi-cel (CD28-based) and tisa-cel (4-1BB-based)-treated B-cell lymphoma patients and advance our understanding on CAR-T resistance. Furthermore, we underscore the need for specific FMC63 epitope detection to deliver information on antigen levels accessible for CD19-CAR-T-cells. Visual abstract

ABSTRACT Viruses have evolved elaborate mechanisms to hijack the host mRNA translation machinery to direct viral protein synthesis. Picornaviruses, whose RNA genomes lack a cap structure, inhibit cap-dependent mRNA translation, and utilize an internal ribosome entry site (IRES) in the RNA 5′-UTR to recruit the 40S ribosomal subunit. IRES activity is stimulated by a set of host proteins termed IRES trans -acting factors (ITAFs). The cellular protein ITAF 45 (also known as PA2G4 and EBP1) was identified as an essential ITAF for foot-and-mouth disease virus (FMDV), with no apparent role in cell-free systems for the closely related viruses harboring similar IRES elements such as encephalomyocarditis virus (EMCV) and Theiler’s murine encephalomyelitis virus (TMEV). Here, we demonstrate that ITAF 45 is a pervasive host factor within cells for picornaviruses containing a Type II IRES. CRISPR/Cas9 knockout of ITAF 45 in several human cell lines conferred resistance to infection with FMDV, EMCV, TMEV, and equine rhinitis A virus (ERAV). We show that ITAF 45 enhances initiation of translation on type II IRESs in cell line models. This is mediated by the C-terminal lysine-rich region of ITAF 45 known to enable binding to viral RNA. These findings challenge previous reports of a unique role for ITAF 45 in FMDV infection, positioning ITAF 45 as a promising antiviral target for various animal viruses and emerging human cardioviruses.

We present an open source, 3D-printed toolbox for avian embryology. The toolbox includes an electroporation chamber for transfecting functional molecular reagents into developing embryos, and a set of live-imaging chambers, which support avian embryo development while presenting them to a wide variety of microscope setups. We demonstrate both electroporation and imaging chambers by transfecting novel fluorescent reporter constructs for the TGF-beta signalling pathway and Pax7, Brachyury, Cdx2 and Sox2:Oct4 transcription factors into chick embryos and performing time-lapse imaging from stages HH3 - HH12 via both widefield and confocal fluorescence microscopy. Open-source code and ready-to-print STL files are freely available from a GitHub repository in line with FAIR (findability, accessibility, interoperability, and reusability) principles.

Cytoplasmic FMRP Interacting Protein 2 (CYFIP2) a component of the Wave Regulatory Complex (WRC), one of the most important players in regulating cellular actin dynamics. Interestingly, CYFIP2 transcript undergoes RNA editing, an epitranscriptomic modification catalysed by ADAR enzymes, that leads adenosine (A) to inosine (I) deamination. CYFIP2 editing in the coding sequence results in a K/E substitution at amino acid 320. The functional meaning of this regulation is still unknown. In this study, we aim at investigating the potential implication of CYFIP2 RNA editing related to actin dynamics during cell differentiation, axon development and synaptogenesis in neural cells. We have generated SH-SY5Y neuroblastoma cell lines in which CYFIP2 gene has been functionally inactivated by CRISPR-Cas9 technology. CYFIP2 KO cells showed profound actin filaments disorganisation and loss of the capability to differentiate into a neuronal-like phenotype. Overexpression of both CYFIP2 unedited (K) and edited (E) isoforms rescued normal capability. Finally, we took advantage of primary neuronal culture where endogenous CYFIP2 was knocked down by shRNA technology and CYFIP2 editing variants were overexpressed. While CYFIP2 KD cells reported a decrease in axon development and spine frequency, CYFIP2-E variants increase the number of axon branches, total axon length and dendritic spine frequency compared to either CYFIP2 KD cells or CYFIP-K variants. Overall, our work reveals for the first time a functional significance of the CYFIP2 K/E RNA editing process in regulating the spreading of neuronal axons during the initial stages of in-vitro development and the process of spinogenesis.

Abstract The CRISPR-associated endonuclease Streptococcus pyogenes Cas9 (SpCas9) enables site-specific DNA cleavage by transitioning from a pre-catalytic conformation to a catalytically active state, yet how its HNH catalytic domain undergoes an approximately 40 Å displacement towards the target DNA has remained elusive. Here, we combined extensive unbiased molecular dynamics simulations, spanning a cumulative timescale of 160 µs, with Markov state modeling to map the kinetic pathway of SpCas9 activation. In vitro DNA cleavage assays and a cellular fluorescence reporter system further validated the atomic-level mechanisms revealed by our simulations. We found that the folding of the L1 linker and unfolding of the L2 linker serve as the principal driving force, inducing a “gear-and-wedge” cooperative motion within the HNH domain. Concurrently, the REC2 domain moved outward to accommodate the displaced HNH domain and formed transient stabilizing interactions with the HNH domain along the activation route. Site-directed mutagenesis of key L2 linker residues and REC2 loops markedly reduced SpCas9 cleavage efficiency in both HEK293T cells and biochemical assays, underscoring their critical role in SpCas9 ribonucleoprotein activation. Collectively, this study provides a high-resolution view of SpCas9 catalytic activation and opens up new avenues for the rational design of SpCas9 variants with enhanced performance and specificity.

The severe acquired respiratory coronavirus–2 (SARS–CoV-2) infection has initiated both acute and chronic COVID–19 disease between 2020 and 2023, currently evolving with other homologous prior coronavirus strains of the Nidoviridae order, which encompasses other prevalent alpha/ beta coronaviruses, but also the Middle East Respiratory Syndrome (MERS-CoV) and SARS-CoV-1, with recent SARS–CoV–2 variants, increasing demands for effective immunogens and therapeutic approaches that will reduce global disease burden and further infection from SARS–CoV-2 affected individuals that may experience post acute sequelae (PASC) or “Long COVID”. Following a worldwide programme of prophylactic vaccination, there is still a dilemma in the efforts to find prophylactic and early therapeutic approaches that would treat novel SARS-CoV-2 variants and prevent future epidemics or pandemics within host human and animal populations, where zoonotic or cross species transfer naturally occurs. Concerns about viral immune escape intersect at a specific point; a gained evolutionary ability of several viruses to co–infect and compete against previous scientific advances since 1796 that remain undetected or asymptomatic during the early stages of infection progressing to symptomatic and severe disease via the double methylation of the 5' end of eukaryotic DNA or RNA-based viral genomes, the 7-MeGpppA2’-O-Me cap, and its double methylation capping process is performed by the activated viral 2’ - O - Methyltransferase (MTase) enzyme, a complex of two viral non-structural proteins (NSPs) joined together through an activation process (NSP10/16) and by N7-Methyltransferase (N7-MTase/NSP14), respectively. Moreover, it was discovered that polymorphic viruses translate NSP1, which prevents the activation of various Pattern Recognition Receptors (PRRs), and consequently, detection of Pathogen-Associated Molecular Patterns (PAMPs) and Damage-Associated Molecular Patterns (DAMPs) alike. NSP1 also silences important interferon-encoding genes (INGs) and interferon-stimulated genes (ISGs), is signalled in a paracrine manner to neighbouring cells, and that induces the apoptosis of host cells, inducing an effect of “trace erase” effect and making the viral infection as immunologically “invisible” as possible during the initial, key stages of viral replication and distribution, all such mechanisms occurring independently of the viruses in cause. Another important viral NSP is NSP14, as it plays two functional roles that are independent of each other; to produce new viral genetic material for the purpose of maintaining the validity of the viral genome as well, and not just transfer a methyl group to the 5’ end of the viral genome. Other viral NSPs share a role with NSP1, 10, 14 and 16 in directly suppressing the activation of PRRs and ISGs, and all such viral proteins help the virus in its process of self-camouflaging against first- and second-line immunity, thereby often severely impacting the quality of the produced adaptive immune responses. The outcome of all such phenomena is the sharp decrease in the host Type I and Type III interferons' (IFNs) rate of synthesis by the host cells, that would usually occur and affect homeostatic cellular pathways, resulting in further viral replication and induced apoptosis. Nonetheless, effects of microbial immune evasion during the development of other viral or carcinogenic pathologies are not widely known. In short, polymorphic viruses developed a proportionate evolutionary response against developed adaptive immune responses, by currently relying on gaps mostly situated in the natural immune system in their process of molecular self-camouflaging. Scientists developed numerous approaches of early treatment that generally showed good success rates and fewer risks of adverse events, and the still early present stages of COVID-19 research should also be taken into consideration whilst filtering for the most appropriate solutions. For example, the administration of recombinant human interferons I and III into the nasal mucosa cellular layer, as key mediators of anti–viral activity, can simulate intracellular infection and stimulate cellular activity in a timely manner, training the innate and adaptive immune system cells to develop and appropriately stimulate an adequate immune response through B and T cells. Another example could involve the treatment of natural and adaptive lymphocytes with a low dose of IFNs I and possibly III, prior to their insertion into the host lymphatic system, possibly alongside additional recruitment of plasmacytoid dendritic cells (pDCs) as further interferon “factories”, all with the purpose of early infection management. It might be that focusing on directly offering the immune system the information about the genetics and protein structure of the pathogen, rather than training its first-line mechanisms to develop faster, excessively increases its specificity, making it reach a level that brings the virus the opportunity to evolve and escape previously-developed host immune mechanisms. It is until the scientific community realises this potentially crucial aspect that large proportions of the world population will probably continue to face serious epidemics and pandemics of respiratory diseases over the coming several decades, evidenced with dengue fever and more recently, monkeypox and possibly avian flu. Of note, it has been indicated that IFN I and / or III display significant immunising, early therapeutic and clinical disease onset-attenuating effects for many other microbial evoked diseases, as well as for a number of oncological diseases. Microbial agents could undergo loss-of-function research upon genes responsible for inducing clinical illness whilst keeping genes responsible for microbial reproduction and transmission at least generally as functional, CRISPR-Cas9 genome editing to have genes encoding proteins suppressive of the host interferon system eliminated prior to human genes encoding Pattern Recognition Receptor activator or agonist proteins, such as outer membrane proteins of Neisseria meningitidis, as well as Type I, Type III and possibly even Type IV Interferons and various ISGs inserted into the microbial genome. Such an approach would be based upon the model of editing genes of harmless bacteria to transform such them into “producers” and “distributors” of human insulin, and could turn several microbial agents into clinically harmless, transmissible “factories” for various key elements of the host interferon system, potentially placing such microbes into a reverse evolutionary path that would be deemed as “natural de-selection”, visibly reducing the average burden of disease and metabolic stresses, which in turn could gradually increase average human and animal lifespans worldwide.

ABSTRACT Gene drives are selfish genetic elements which promise to be powerful tools in the fight against vector-borne diseases such as malaria. We previously proposed population replacement gene drives designed to better withstand the evolution of resistance by homing through haplolethal loci. Because most mutations in the wild-type allele that would otherwise confer resistance are lethal, only successful drive homing permits the cell to survive. Here we outline the development and characterization of two ΦC31-Recombination mediated cassette exchange (RMCE) gene drive docking lines with these features in Anopheles gambiae , a first step towards construction of robust gene drives in this important malaria vector. We outline adaption of the technique HACK (Homology Assisted CRISPR knockin) to knock-in two docking site sequences into a paired haplolethal-haplosufficient (Ribosome-Proteasome) locus, and confirm that these docking lines permit insertion of drive-relevant transgenes. We report the first anopheline proteasome knockouts, and identify ribosome mutants that reveal a major hurdle that such designs must overcome to develop robust drives in the future. Although we do not achieve drive, this work provides a new tool for constructing future evolution-robust drive systems and reveals critical challenges that must be overcome for future development of gene drives designed to target haplolethal loci in anophelines and, potentially, other metazoans.