Abstract Drug resistance to multiple tyrosine kinase inhibitors (TKIs) is a major issue during clinical management of hepatocellular carcinoma (HCC). Here, through multiplexed in vitro and in vivo CRISPR knockout and base editing screens, we elucidate the resistance mechanisms of HCC to typical TKIs (sorafenib, lenvatinib and regorafenib). Multiple genetic or epigenetic alterations can drive resistance to these TKIs through divergent mechanisms. Among them, a tumor cell-intrinsic (rather than tumor microenvironment-driven) extracellular matrix remodeling mechanism stands out across multiple resistance models and clinical samples. Using druggable gene CRISPR knockout screens, we identify proteasome as a prominent and convergent vulnerability of multiple TKI-resistant HCC cells. We further validate the efficacy of clinically available proteasome inhibitor bortezomib in treating TKI-resistant HCC in multiple preclinical models. Mechanistically, increased p21 expression and dysregulated proteolytic machineries in TKI-resistant tumors account for the pronounced sensitivity to bortezomib. Our work not only delineates a comprehensive landscape of drug resistance mechanisms in TKI-treated HCC, but also suggests actionable therapeutics with immediate clinical potential against these tumors.

Throughout several centuries, infectious pathogenic agents have been used as models for the ongoing efforts of vaccine development, which saved hundreds of millions of lives from life-threatening infectious diseases worldwide. Nonetheless, there has been a missing gap that various polymorphic microbes have been taking advantage of in their evolutionary pathway: the interferon system, which often prevented the timely activation of second and third-line host immunity, leading to chaotic and mismatching immune responses. The phenomenon of increased incubation period of various infectious diseases may be a result of the increased abilities of such microbial agents to directly and indirectly undergo molecular self-camouflaging, which prevents the activation of Type I and Type III Interferon-encoding genes (INGs) in indirect and direct manners respectively, and cleaves the mRNA molecules encoding such interferon glycoproteins, often causing major delays in the process of autocrine and paracrine signalling of Type I and Type III Interferon glycoproteins, which in turn allows an unrestricted, exponential increase of the microbial load/count, giving rise to a statistical probability that the quality of the delayed immune response will be low and contributory to the processes of pathogenesis and pathophysiology. Some microbial proteins as such also inhibit the translation of Interferon-Stimulated Genes, thereby substantially affecting the signalling rates within the cytokine system and often bringing a negative domino effect upon the activation rates of the adaptive immune system. Apprehending the foundational layer of the current problems in evolutionary microbiology, epidemiology and public health studies is most likely crucial for the course of immunological, pharmaceutical and vaccine-related clinical research. In the current case, it is the complex set of molecular capabilities to suppress Type I and Type III Interferon-based signalling displayed by several polymorphic microbes of public health concern, and it may be that the rates of immunopathogenesis induced by such microbes are directly proportional with such pathogenic abilities of induced interferon suppression. Proportional medical responses could include the development of approaches involving low dosages of human recombinant Type I and Type III Interferon glycoprotein and perhaps also of protollin in the nasopharyngeal cavity, potentially bringing an example of putting a novel concept of a “United Immune System” into practice. Furthermore, similar dosages of such interferons could be administered into human immune cells including plasmacytoid dendritic cells, as well as natural and adaptive lymphocytes, to optimise their immune function and integrity against various environmental hazards. Ultimately, clinical researchers may isolate the pathogenic agents, attenuate them through the process of loss-of-function laboratory research, before performing gene editing to insert Type I, Type III and perhaps also Type IV Interferon-encoding, perhaps as well as Pattern Recognition Receptor (PRR) Agonist-encoding genes that specifically match the PRR targeted by the implicated microbes, into their genomic profile and potentially releasing the genetically-modified pathogens back into the environment transmissible factories of Type I and Type III Interferons, perhaps as well as of specific PRR Agonist proteins, which could include outer membrane proteins from the B serogroup of Neisseria meningitidis bacteria. If the microbial genetic activities implicating evasion of the interferon system are too intense and multilateral, at least some of the microbial genes responsible for such activity could be permanently removed in some exchange with the human genes encoding major elements of the interferon system that would be inserted into the microbial genome afterward. While the biological mechanisms discussed below are grounded in published interferon and immune-evasion literature, the present manuscript does not assert practical feasibility of transmissible vaccine strategies. Rather, it evaluates whether theoretical epidemiological and evolutionary models can define mathematical upper and lower bounds on such a concept under extreme and idealised assumptions. The objective is to test the internal coherence of the framework, not to imply translational readiness. It may be important to mention that the process of clinical weakening of the isolated microbes would be aimed at reducing the activity of microbial genes implicated in pathogenesis and pathophysiology, and perhaps not as much microbial genes involved in reproduction and transmission. Such a change may bring various pathogenic agents into a path of evolutionary self-destruction, as they would start producing and sending signals to the proximal, innate immune system as soon as they enter the first host cells, making their same processes of induced innate immune suppression ineffective, and several dilemmas in microbial evolution could ultimately be tackled as a result, possibly even at least attenuating the phenomenon of acquired antibiotic resistance by various pathogenic bacteria. A clinical approach as such is likely based on the model of increasing the accessibility to insulin-based treatment against Diabetes Mellitus via insulin-encoding gene insertion into the genomes of harmless bacteria prior to their administration into human host organisms, which saved millions of lives worldwide. Processes of shrinkage of any level of limitations to potential efficacy would include the manual utilisation of inhalators, oral drops and/or injectable serums containing such modified microbes to ensure that such an immunising effect would be conferred simultaneously with exposure to the artificially-changed genetic version of the microbe, effectively creating an “active evolutionary trap” for the pathogens, potentially resulting in their gradual de-selection whilst they continue to transmit just sufficiently enough to produce lasting immune memory. In other words, a phenomenon of “pathogen baptism” could occur, implicating a domination of “domestic variants” over wild-type variants in the environment, with the former becoming like “wild animals”, as they would remain the only virulent pathogenic variants and gradually even become extinct, with the “domestic” variants becoming dominant, according to the viral quasispecies theory. This set of clinical responses, including targeted immunoediting and gene vector strategies, can be analogized to a strategic operation against a mega-hurricane. The immune system, overwhelmed by storm-like chaos, cannot function effectively from the outside. Thus, medical intervention must act like military aircraft entering the eye of the storm from above – where calm resides – not to be engulfed, but to deploy stabilizing agents from within the calm zone. Only then can the storm’s structure be undone without triggering systemic devastation. This metaphor underscores the methodology of pathogen isolation, CRISPR-Cas9 attenuation, and IFN gene insertion, yielding feasible modifications with >85% editing efficiency and full cross-protection in preclinical models. Such a metaphor could potentially be informally regarded as a conquest from within, while remaining of another world). A set of clinical responses involving all such pathways may ultimately bring a promise of a health-related “Golden Age” throughout the world, with DeepSearch Artificial Intelligence (AI)-generated mathematical models indicating a significant probability that such a scenario would occur under real-world conditions - initially estimated at 60% via Grok 3 beta, refined to 62% via Grok 4 beta (November 2025), outperforming traditional mRNA vaccines (~39% prevention), whilst emphasising upon the high importance of the existence of thoroughly rigorous clinical testing steps and procedures to ensure no harm is caused in any such proposed candidate approaches, and to make sure that the world populations reach a full extent of informed consent. Finally, to concisely bridge from blueprint to prototype for conceptual, hypothetical purposes, we outlined a phase progression: (1) Introduction of PoC protocols to the present study, (2) In-Vitro Proof-of-Concept (1 - 3 months) - transfecting HEK293/Vero cells with CRISPR-edited IFN cassettes, targeting >90% efficiency and 104 IU / mL secretion via ELISA/WB - (3) Animal Validation (2 - 4 months) - Nasal-dose of hACE2 mice (n = 25/group), assessing 80-100% cross-protection and <0.1% reversion - (4) Iteration - recalibrating SEIR models with empirical data (e.g. β_d = 0.85), elevating projections to 68% pandemic prevention. Such a roadmap, aligned with CEPI/NIH accelerators, ensures ethical LOF-only prototyping, de-risking deployment whilst fostering a “United Immune System” concept for global resilience. Under more constrained assumptions, upper-bound theoretical estimates suggested evolutionary stability approaching 99.2%, whilst conservative stress-tested scenarios yielded considerably lower estimates (~80–85%). These values represent theoretical model-dependent bounds, and not empirical guarantees. This study presents a theoretical evolutionary modelling framework and does not advocate real-world deployment of transmissible agents.

Abstract The commercial deployment of genome-edited crops is frequently bottlenecked by the extended juvenile phases of perennial species and the complex regulatory landscapes governing plants with integrated exogenous DNA. Speed breeding protocols, which utilize environmental manipulation to accelerate development, have proven effective for annual cereals but often lack efficacy in perennials. In this study, we report a genetic speed breeding system that couples the overexpression of the floral integrator FLOWERING LOCUS T ( FT ) with CRISPR/Cas9-mediated genome editing. Using Nicotiana tabacum as a model for polyploid crops, we demonstrate that constitutive expression of Arabidopsis thaliana FT ( AtFT ) reduces the generation time from 12 weeks to approximately 3 weeks without compromising fertility or seed viability. To leverage this acceleration for trait improvement, we engineered a binary vector co-expressing AtFT and a CRISPR/Cas9 cassette targeting the NtDFR ( dihydroflavonol 4-reductase ) loci. This integrated system induced rapid flowering and simultaneous disruption of anthocyanin biosynthesis, yielding ntdfr mutants with a distinct white-flower phenotype. Importantly, the edited alleles were heritable, while the FT-Cas9 transgene could be segregated out in the next generation, restoring the wild-type photoperiodic response in the edited progeny. This transgenic facilitator approach offers a scalable platform for the rapid introgression of edited traits into recalcitrant crop species, potentially reducing the breeding cycle of perennials from years to months.

Abstract Background Earliness of tuberisation is an important agronomic trait. It was demonstrated earlier that GIGANTEA (GI), a plant-specific nuclear protein that regulates multiple processes, is indirectly involved in tuberisation in a diploid potato. Commercial potatoes, including the cultivar Désirée, are tetraploids and carry two copies of GI genes, designated GI.04 and GI.12 . The aim of our study was to explore the role of the two GI genes in Désirée in relation to tuberisation. Results To obtain information on GI.04 and GI.12 functions in Désirée, mutations were introduced into the two genes individually and simultaneously using the CRISPR/Cas9 system. Two different segments of the genes were targeted by gRNAs. PCR was used for mutant identification. Three mutants from each mutagenesis were selected, and the mutations were localised at the DNA sequence level. The phenotype and tuberisation of the plants were tested by growing the plants in pots in a greenhouse. The individual mutations affecting all four copies of the genes, in general, reduced plant size. Plants of one GI.04 mutant line and two GI.12 mutant lines with truncated proteins and deletions in the 816–869 and 834–863 amino acid (a.a.) regions, respectively, were shorter and remained green for a longer time than Désirée. GI.04 and GI.12 mutants with truncation or deletion in the 567–632 a.a. and 618–694 a.a. regions, respectively, differ in phenotype; one GI.04 mutant had longer, whereas all three GI.12 mutants and the double mutants had shorter life cycles. However, only one of the GI.12 mutants and one of the double mutants tuberised earlier than Désirée. The tuber yield of the double mutant with the shortest life time was lower than that of Désirée. Conclusions Both GI genes of Désirée influence the development and life cycle length of plants. The influence of GI.12 is more pronounced than the influence of GI.04. In conjunction with the shortened lifetime, the onset of tuberisation occurs earlier.

Abstract The mechanical regulation of nuclear volume is a fundamental yet under-explored aspect of embryogenesis. We developed an automated computational framework in Python to quantify 3D nuclear morphometry in zebrafish (Danio rerio) embryos. Applying this pipeline to cdh2-CRISPR mutant image stacks from the BioImage Archive (Accession: S-BIAD1405), we observed pronounced nuclear hypertrophy associated with reduced cadherin-mediated adhesion. Using voxel scaling factors verified in ImageJ (pixel width/height = 0.991492 µm; z spacing = 1.0 µm; consistent across WT and mutant stacks), we observed a ≈12.0% increase in median nuclear volume (1,606 vs. 1,434 voxels; Mann–Whitney U, p = 0.0407), while nearest-neighbor distances remained broadly similar, suggesting that local packing is comparatively preserved. These results support a model in which cadherin-dependent mechanical coupling contributes to nuclear size homeostasis. We provide biological insight into the mechanobiological role of cdh2 and an open-source workflow for reproducible volumetric analysis in complex 3D biological systems.

Purpose: The aim of the study is to enable health professionals, researchers, and organizations, to learn about the most advanced technological innovations reported in the medical literature. Methods Content analysis (CA) was used to gather data by expert investigators from “ScienceDirect” database from 2018 to 2022 inclusively. Megastat was used to analyze the frequency counts of the reported key. Results A total of N= 10,767 data point related to technological innovation in medicine were identified. Frequency counts and data interactions revealed three major categories, innovative domains, information technology, and medicine, each defined by their most frequently cited terms: precision medicine (n=1448) and regenerative medicine (n=1242), AI (n=1797) and telemedicine (n=1114), and messenger RiboNucleic Acid (mRNA) (n=1008), cancer immunotherapy (n=712), and CRISPR (n=620), respectively. Conclusions The CA indicates that the literature reports the medical community and healthcare industry as actively engaging with various technologies to advance patient care and enhance quality of life. Precision medicine, regenerative medicine, AI, telemedicine, mRNA, CRISPR, and cancer immunotherapy were the most frequently cited. Future studies could expand the scope by including additional databases to provide a more comprehensive overview.

Malaria, caused by Plasmodium parasites, remains a global health crisis, necessitating novel therapeutic strategies targeting host-parasite interactions. During liver stage in-fection, parasites exploit host vesicular trafficking machinery, particularly SNARE pro-teins that mediate membrane fusion. Using a CRISPR/Cas9 knockout system in HeLa cells combined with advanced microscopy of Plasmodium berghei-infected HeLa cells, we identified specific endolysosomal SNAREs VAMP7, VAMP8, Vti1B, and Stx7 to be re-cruited to the parasitophorous vacuole membrane (PVM) with distinct temporal profiles. This demonstrates the parasite’s precise manipulation of host endolysosomal trafficking pathways. VAMP7 and Vti1B localized to the PVM within 30 minutes post-infection suggesting potential roles during invasion, while VAMP8 and Stx7 appeared later toward 24 hpi, coinciding with increased nutrient acquisition. Single gene deletions showed minimal impact, but combinatorial knockouts revealed critical redundancy. VAMP7-VAMP8 as well as VAMP7–Vti1B double KO significantly reduced parasite in-fection and growth, with Vti1B playing a dominant role. Triple KO phenotypes mirrored VAMP7-Vti1B disruption, underscoring Vti1B’s dominant role. SNARE depletion also impaired lysosome-PVM association and LAMP1 positive vesicle recruitment. Our findings indicate Plasmodium hijacks a coordinated host SNARE network to fuse lysosomes with the PVM for nutrient uptake. Targeting Vti1B-containing complexes disrupts this pathway without host cell toxicity, offering a promising host-directed antimalarial ap-proach.

Cancer immunotherapy holds immense potential for the future of medicine within the study of cancer therapeutics; however, most therapies are undermined by T-cell exhaustion and tumor immune evasion. T-cell exhaustion is caused by chronic antigen stimulation in the tumor microenvironment (TME), leading to dysfunctional epigenetically enforced states by transcription factors such as TOX and the NR4A family. Simultaneously, tumors evade the immune system by silencing MHC-1 molecules. Proposed is a synergistic approach that addresses these obstacles, involving the engineering of TCR-T cells for enhanced durability against the TME through CRISPR-Cas9-mediated knockout of exhaustion transcription factors, and the reprogramming of cancer cell transcriptomes using DNA methyltransferase (DNMTi) and histone deacetylase (HDACi) inhibitors. Furthermore, this review additionally incorporates: metabolic resistance, addressing the transfer of mitochondria from neurons to cancer cells, and enhancing oxidative phosphorylation (OXPHOS). These are proposed interventions for these obstacles with specific biomarkers (T-cell signatures, tumor epigenetic landscape, and tumor innervation density). By combining exhaustion-resistant T-cells with a reprogrammed tumor that is visible to the immune system, there is potential to overcome immune resistance and improve therapeutic outcomes for patients.

Advances in technology have provided a better understanding of the genetic basis of neurodegenerative disorders and their underlying molecular pathophysiology. However, treating these disorders with conventional strategies is a major challenge. The approval of gene-targeted therapy for spinal muscular atrophy (SMA) has laid the foundation for developing therapies for other neurodegenerative disorders. Highly personalized gene therapy trials have been reported. As intensive research and efforts to advance gene-targeted therapies continue, this review provides an overview of viral and non-viral vectors and delivery methods, as well as treatment strategies, including gene addition, replacement, editing, silencing, and splice modulation. Gene-targeted approaches and clinical trials for SMA and amyotrophic lateral sclerosis (ALS) have demonstrated success, and additional studies are in progress. The design of efficient clinical trials which facilitate successful translation into clinical practice is of critical importance. Key considerations include the selection of appropriate disease models, understanding the natural history of the disease, and establishing well-defined outcome measures to assess prognosis of the disease and therapeutic efficacy. Finally, the precision of CRISPR-gene editing may facilitate the development of new therapies.

Abstract Drought stress severely limits grape productivity. Gibberellin (GA) metabolism is involved in drought responses, yet the role of specific GA catabolic genes remains unclear. In the present study, VvGA2ox8-Like, a member of the grape GA 2-β-dioxygenase (VvGA2ox) gene family, was identified to confer drought tolerance in Vitis vinifera cv. ‘Pinot Noir’. As a GA catabolic enzyme, VvGA2ox8-Like expression is significantly induced by drought stress. Transient transformation of ‘Pinot Noir’ grape leaves showed that VvGA2ox8-Like overexpression decreased H₂O₂ and MDA contents, while increasing proline content, antioxidant enzyme activities, and drought-responsive gene expressions; in contrast, virus-induced gene silencing (VIGS) of VvGA2ox8-Like exhibited opposite results. Furthermore, VvGA2ox8-Like overexpression in ‘Pinot Noir’ grape calli further confirmed its positive regulatory role in drought tolerance, whereas CRISPR-Cas9-edited VvGA2ox8-Like calli showed opposite phenotypes. A drought-responsive protein VvGRX-S7 was identified as an interacting partner of VvGA2ox8-Like via yeast two-hybrid (Y2H) assay. RNA-sequencing (RNA-seq) analysis revealed that VvGA2ox8-Like overexpression significantly affected the biosynthesis of secondary metabolites, phenylpropanoids, and flavonoids. In conclusion, VvGA2ox8-Like plays a key role in grape drought tolerance, providing theoretical support for elucidating the molecular mechanism of grape drought resistance.

Abstract Background: Antimicrobial resistance (AMR) is a mounting global threat to human, animal, and environmental health. Low- and middle-income countries (LMICs) bear a disproportionate burden due to limited diagnostics, weak regulatory frameworks, and constrained access to novel antibiotics. Conventional therapies are increasingly ineffective, underscoring the urgent need for innovative, precision-targeted interventions. Scope: This narrative review synthesizes emerging next-generation antimicrobial strategies—including bacteriophage therapy, CRISPR-Cas antimicrobials, engineered antimicrobial peptides (AMPs), enzybiotics, and nanotechnology-enabled delivery systems—through a One Health lens. Emphasis is placed on feasibility, scalability, and applicability in LMIC contexts. Key Findings: Preclinical and early clinical studies demonstrate that phages, CRISPR-Cas antimicrobials, and engineered AMPs can reduce multidrug-resistant infections by 60–95%. Enzybiotics and nanotechnology platforms enhance biofilm disruption, stability, and targeted delivery. Combinatorial approaches (e.g., phage–CRISPR, AMP–nanoparticle formulations) further improve antimicrobial efficacy and may mitigate resistance development. Challenges & Outlook: Deployment in LMICs is constrained by delivery optimization, manufacturing costs, regulatory gaps, and infrastructure limitations. Solutions tailored to local production capacity, cold-chain independence, and cost-effectiveness are critical. Integrating these strategies with genomic surveillance, stewardship programs, and One Health governance can accelerate safe and equitable implementation. Tailoring next-generation antimicrobials to LMICs requires cost-effective, locally producible, cold-chain-independent formulations, integrated One Health deployment, and strengthened regulatory and workforce capacity to ensure equitable access and sustainability. Conclusion: Next-generation antimicrobials provide precision-targeted, multi-domain solutions to combat AMR. Strategic combinations, optimized delivery platforms, and LMIC-adapted policies are essential to translating preclinical promise into effective One Health interventions that reduce the global AMR burden.

Abstract The organization of signaling proteins into multi-component complexes is essential for innate immune responses and is often achieved through the oligomerization of death domains (DDs). The Myddosome, composed of MyD88 and IRAK family kinases, exemplifies this mechanism. However, how DDs assemble in an ordered and regulated manner to form Myddosomes to control signaling output remains unknown. Here, we show that IRAK4 kinase activity and autophosphorylation are required for IRAK4:IRAK1 heterotypic DD interactions. We discovered that IRAK1 is autoinhibited by its kinase domain, and IRAK4 phosphorylation relieves this inhibition, enabling IRAK1 DD incorporation into Myddosomes. This phosphorylation-dependent switch similarly controls IRAK2/3 incorporation. Thus, IRAK4 autophosphorylation acts as an energy-dependent molecular switch that integrates phosphorylation with protein oligomerization to drive Myddosome maturation and signal transduction. This mechanism demonstrates how enzymatic activity can impose temporal and spatial control over macromolecular assembly to ensure directional and context-dependent inflammatory signaling.

Abstract Hereditary Myopathy with Early Respiratory Failure (HMERF) is a progressive titinopathy caused by dominant missense mutations in the A-band region of TTN , a domain essential for sarcomere stability. Patients suffering with HMERF manifest muscle weakness, early respiratory involvement, and reduced life expectancy, yet no effective therapies currently exist. A major barrier has been the lack of an animal model that replicated HMERF pathology. In this study, we established the first CRISPR-Cas9 engineered mouse model of HMERF, carrying a patient-derived missense mutation in Ttn . Homozygous mutant mice exhibited a severe and uniform phenotype, including kyphosis, thoracic deformities, abnormal gait, diaphragm weakness, and premature death. Histological analysis revealed disrupted sarcomeres, abnormal myotilin accumulation, and necklace-like cytoplasmic bodies in diaphragm muscle, resembling human pathology. Multi-omics approach revealed consistent dysregulation of genes and proteins linked to muscle structure, cytoskeletal integrity, and cellular homeostasis, representing disease pathomechanisms. A major limitation of this study was the restricted availability of muscle tissue, which prevented broader analysis across multiple muscle types. Nevertheless, overlapping transcriptomic and proteomic dysregulation, including differential splicing, highlight key molecular effects driving disease progression. In conclusion, this mouse model provides mechanistic insight into HMERF and establishes a platform for evaluating therapeutic strategies. It represents an essential step toward developing targeted interventions for this rare and severe neuromuscular disorder.

Abstract In the present study we have generated crickets with knockout of either per or tim gene by CRISPR/Cas9 based genome-editing. We also obtained naturally occurred per − mutant lacking a large coding region including PAS domains. To investigate synergistic effects, a per − and tim KO double mutant was produced by applying genome editing to the per − crickets. Under constant darkness (DD), tim KO crickets showed a locomotor rhythm with a free-running period significantly shorter than that of the parental strain. The per KO and per − crickets showed basically similar phenotype of locomotor rhythm: they exhibited arrhythmic pattern first two to three weeks after transfer to DD but subsequently showed a complex rhythmic pattern with a single or multiple components with significantly longer free-running periods. In the per − ; tim KO double mutants, approximately 60% of individuals became arrhythmic, while the remaining 40% exhibited complex rhythm with extremely longer free-running periods under DD. These results suggest the existence of underlying oscillatory mechanism that is responsible for regulating locomotor rhythms independently of the canonical per/tim feedback loop. Furthermore, we generated per − reporter line with egfp knocked in exon 1 of the per gene. EGFP expression was detected in three distinct clusters of cells within the optic lobe: two located along the dorsal and ventral boundaries between the lamina and medulla neuropils, and one situated in the proximal medulla neuropil near the accessory medulla. These findings suggest that these specific cells constitute a circadian clock network that governs circadian locomotor rhythms.

Abstract The growing world-wide population and climate-induced agricultural setbacks demand innovative approaches to sustainable food production. Hydroponic systems offer promising solutions through resource-efficient, soilless cultivation methods suitable for urban and drought-prone regions. However, the build-up of organic matter in recirculating nutrient solutions elevates biochemical oxygen demand (BOD), leading to dissolved oxygen depletion, disrupted microbial balance, compromised plant health, and potential food safety risks through pathogen proliferation. This review examines synthetic biology as a strategy for optimising BOD degradation in hydroponic systems. We explore the application of genetically engineered microorganisms, including Bacillus subtilis , Pseudomonas putida , and Rhodococcus species, equipped with enhanced catabolic pathways for targeted organic matter degradation. Advanced genetic tools such as CRISPR-Cas9 gene editing, metabolic pathway engineering, and synthetic microbial consortia design are evaluated for their efficacy in maintaining water quality while supporting crop productivity. The integration of biosensor technologies, Internet of Things (IoT) platforms, and real-time monitoring systems allows for dynamic, feedback-responsive bioremediation strategies. Comparative assessments demonstrate synthetic biology's benefits over traditional BOD management methods in terms of specificity, energy efficiency, adaptability, and environmental sustainability. We address biosafety mechanisms (kill switches, auxotrophy), regulatory frameworks, ethical implications, and public acceptance challenges. This review highlights successful pilot implementations, discusses scalability for commercial operations, and identifies future research directions, emphasising interdisciplinary approaches, long-term ecological impact assessments, and cost-effective designs for small-scale farmers. Ultimately, synthetic biology-based BOD optimisation offers a strategic pathway toward resilient, sustainable, and safe hydroponic food production systems that contribute to global food security.

Abstract The histone methyltransferase SETD2 and its associated histone mark H3 lysine 36 trimethylation (H3K36me3) are frequently lost in cancer, identifying SETD2 tumour suppressor loss as an important therapeutic target. Here we show that SETD2-deficient cancer cells are profoundly sensitive to RITA (2,5-bis[5-hydroxymethyl-2-thienyl] furan; NSC652287). Exposure of SETD2-deficient cancer cells to RITA results in significant p53 induction and apoptosis. However, TP53-deficient cells also exhibit RITA sensitivity suggesting p53 induction is an effect rather than a cause of RITA sensitivity. We find that RITA sensitivity is dependent on the phenol sulfotransferase SULT1A1, which is highly upregulated in SETD2-deficient cells. Accordingly, structural modifications of RITA, predicted to compromise its sulfation, ablated its activity. Further, SETD2-deficient cells can be targeted with YC-1, another SULT1A1-dependent anti-cancer agent. RITA sensitivity was associated with defects in DNA replication, leading to delays in S-phase progression, increased recruitment of replication stress markers, and reduced replication fork progression. Consistent with this, global target deconvolution using thermal profiling (2D-TPP) identified a broad range of RITA target proteins, including many involved in DNA replication stress. Together, these findings support the exploitation of SULT1A1 expression as a novel therapeutic strategy to target SETD2-deficient cancers.

Abstract Public support for gene editing, particularly for therapeutic purposes, remains strong. Recently, the Ministry of Health in Saudi Arabia approved CRISPR-Cas9 for treating Sickle Cell Disease and beta thalassemia. This study aims to assess the Taif population’s opinion on gene editing and their knowledge of genetic modification. In this cross-sectional study, a questionnaire was distributed online from March 2, 2024, to June 15, 2024, to 747 residents of Taif City aged 18 and older. Among the respondents, 14.7% reported that they or their family members suffer from a hereditary disease, and 65.7% either work or study in the healthcare field or have a family member involved in healthcare. Additionally, 50.7% had previously heard of genetic modification. Marital status, number of children, and education level did not significantly influence opinions on genetic editing, whereas affiliation with the healthcare field was significantly associated with greater acceptance (p = 0.023), while a family history of hereditary disease showed a trend toward significance (p = 0.055). Public opinion strongly supports using genetic editing to treat life-threatening diseases in adults and embryos (63.2% and 73.6%, respectively). However, opinions are more divided on non-disease traits. Many respondents expressed interest in enhancing intelligence (73.8%) and strength (75.8%), as well as altering height (67.8%) and hair color (60.7%). While support was strong for therapeutic use, opinions were divided on enhancement, reflecting ethical tension despite high interest in modifying non-disease traits. Notably, 51.4% believed that using genetic editing for non-medical purposes crosses ethical boundaries and exceeds nature’s limits. Awareness of gene-editing techniques was not significantly associated with acceptance (p = 0.108). In conclusion, public acceptance of gene editing in Taif is high, particularly among healthcare-affiliated individuals. Increasing public awareness remains essential to bridge ethical concerns and support informed engagement.

Abstract Innate antiviral factors are critical components of host defense. However, many physiologically relevant mechanisms restricting HIV-1 replication remain to be identified. To uncover such factors, we developed an HIV-1–guided CRISPR-Cas9 screen using replication-competent HIV-1 libraries encoding 77,441 distinct sgRNAs targeting all annotated human genes. Viral propagation in the presence or absence of IFN-β enabled the identification of multiple cellular genes that significantly restrict HIV-1 replication. Among the top hits were factors involved in DNA metabolism and repair (DNase1L2, NTHL1, POLE4, RMI2), chromatin regulation (MPND), and innate immune signaling (GNB1L, TRIM9). Their restrictive effects were validated in primary CD4⁺ T cells, confirming relevance in HIV-1’s major target cells in vivo. Together, these findings establish genome-wide replication-competent HIV-1 CRISPR screening as a powerful approach to identify restriction factors and uncover a previously unrecognized role of genome integrity pathways in cell-intrinsic antiviral immunity.

Abstract Titin, the largest muscle protein, plays a key role in the architecture of sarcomeres in both the heart and skeletal muscles. Due to its crucial role, variants in this gene have a critical impact on human health. Titinopathies include severe cardiomyopathies and dominant and recessive skeletal muscle diseases, associated with several pathogenic variants. Among these, titin A150/FN3-119 domain variants are associated with hereditary myopathy with early respiratory failure (HMERF), a life-threatening disorder characterized by respiratory failure and proximodistal muscle weakness. Although murine and fish models have been developed for a wide range of titinopathies, an HMERF model is lacking. Here, we generated and characterized an HMERF knock-in model using Oryzias latipes (medaka fish). Upon the generation of this model, which carries the most common HMERF missense variant (p.C31712R), we found that the mutants had impaired muscle structure, with homozygous larvae exhibiting a more severe phenotype than their heterozygous siblings. Focusing our study on the homozygous larvae, we performed RNA sequencing (RNA-seq) analysis, revealing significant dysregulation of genes with key roles in muscle filament organization and autophagy pathway. This suggests exacerbated muscle damage and dysfunction. These results were corroborated by locomotor analyses and mechanical studies, which revealed that homozygous larvae exhibit limited movement and reduced muscle fiber capability to generate force and shortening at high speed. These results demonstrate that structural abnormalities directly correlate with the impaired function in HMERF mutants. Taken together, the altered muscle structure, impaired locomotor behavior, and dysregulated gene expression underscore the complex pathological mechanisms underlying HMERF disease. Beyond elucidating HMERF-disease mechanisms, our work highlights the value of genome editing in medaka fish, a powerful and versatile model system to dissect the molecular basis of human muscle diseases.

Abstract Viruses are the most pervasive biological entities on Earth and they profoundly shape host ecology and evolution. However, for many microbial lineages, knowledge of their viromes remains limited, especially for those inhabiting remote environments, including deep-sea ecosystems. Here, we leverage one of the most extensively cultivated and genomically characterized archaeal lineages, the Thermococcales, to identify novel viral genomes. By utilizing CRISPR spacers from isolates and spacer arrays reconstructed from metagenomes, we mined mobile genetic elements (MGEs) in 1,172 publicly available and newly sequenced hydrothermal vent metagenomic datasets. Comparative genomics and identification of viral hallmark proteins revealed 620 viral genomes across 19 taxonomic families, most of which were previously undescribed. Structural modeling of major capsid proteins (MCPs) revealed diverse virion morphotypes, including viruses with spindle-shaped, head-tailed, icosahedral, filamentous, ovoid and bacilliform virions, greatly expanding the previously limited Thermococcales virome. Family-level comparisons uncovered extensive flux of virus-encoded replication proteins that are evolutionarily and structurally distinct from host homologs, as well as dramatic variation in glycan-binding lectins suggestive of diverse infection strategies. Together, our results substantially broaden the Thermococcales virosphere and demonstrate the power of combining cultivated isolates with culture-independent, CRISPR-guided metagenomics to interrogate archaeal virus diversity and evolution.

Background: NOTCH receptors play a pivotal role in carcinogenesis. Upon ligand binding, a cascade of proteolytic cleavages mediated by ADAM proteases and the γ‑secretase complex activates the receptor, ultimately releasing the NOTCH intracellular domain (NICD). NICD translocates to the nucleus, where it regulates gene expression. This review mainly aims to evaluate γ‑secretase inhibitors (GSIs) as anticancer agents in preclinical and clinical settings, with a focus on their ability to block tumor progression, target cancer stem cells, and overcome resistance to standard therapies. Methods. A systematic search was conducted in the ISI Web of Science, PubMed, and Scopus databases, following PRISMA guidelines. The review included preclinical in vitro and in vivo studies, as well as clinical trials, investigating GSIs, either as monotherapy or in combination with other treatments, in TNBC, metastatic melanoma, PDAC, gastric cancer, and NSCLC. Exclusion criteria included duplicates, non‑English articles, studies published before 2010, studies on non‑cancer conditions, research unrelated to NOTCH signaling, and studies outside the selected cancer types. 69 articles were included and categorized into the five types of cancer analyzed (20 on NSCLC, 22 on TNBC, 11 on metastatic melanoma, 7 on GC, and 9 on PDAC). Of these, 60 studies correspond to preclinical research in the types of cancer and 9 studies correspond to clinical trials in the types of cancer except for GC (Figure 7). Two independent authors screened and extracted relevant data, with disagreements resolved by the corresponding author. Findings were synthesized qualitatively across cancer types under study. Results. This review summarizes therapeutic advances involving GSIs in cancers driven by oncogenic NOTCH signaling, based on the 69 articles included. Preclinical studies show that GSIs synergize with chemotherapy and radiotherapy, particularly in NSCLC, melanoma, and TNBC, and block EMT, overcome therapeutic resistance, and improve prognosis. Commonly used GSIs include DAPT and RO4929097, which enhance the efficacy of agents such as gemcitabine (PDAC), paclitaxel, osimertinib, erlotinib, and crizotinib (NSCLC), and 5‑FU (gastric cancer, TNBC). Promising strategies include combining GSIs with SAHA, ATRA, CB‑103, and other NOTCH signaling targeting molecules, either alone or with chemo‑ and radiotherapy. Clinical trials with GSIs, however, remain limited. RO4929097 is the most extensively tested GSI in clinical settings. PDAC trials combining GSIs with gemcitabine showed no benefit; melanoma trials yielded modest outcomes; and TNBC trials demonstrated partial responses to GSIs but overall low efficacy and significant adverse events. Discussion and Conclusion. Despite encouraging preclinical evidence, clinical trials with GSIs have underperformed, largely due to tumor heterogeneity, dosing limitations, and the non‑selective nature of γ‑secretase inhibition. Other NOTCH inhibitors such as DLL4 antibodies also resulted in partial responses and secondary effects. Future strategies should prioritize receptor‑specific NOTCH inhibitors, patient stratification based on NOTCH pathway activation, and optimized combination regimens. Emerging approaches include integrating immunotherapy with advanced technologies such as CRISPR, CAR‑T cells, and bispecific antibodies, as well as targeted delivery systems to enhance efficacy and reduce toxicity. Additional research directions include addressing the tumor microenvironment and EMT‑driven resistance, elucidating mechanisms of immune evasion, and inhibiting tumor angiogenesis. Finally, leveraging artificial intelligence and big‑data‑driven personalized medicine, including sex‑specific considerations, will be essential for improving patient outcomes.

Abstract CRISPR/Cas9 genome editing provides a powerful framework for interrogating gene function in honey bees (Apis mellifera). Yet, empirical application remains challenging due to biological constraints, including haplodiploid genetics, narrow embryonic injection window, and the social rearing requirements that complicate functional validation. These constraints necessitate in silico pre-screening to maximize editing success before resource-intensive wet-lab implementation. Within the omnigenic framework, which distinguishes core regulatory genes from peripheral loci buffered by network effects, vitellogenin ( Vg ) represents an optimal target which ancestrally dedicated to yolk provisioning, it has been co-opted to orchestrate diverse non-reproductive functions including longevity, stress resistance, immunity, and social behavior. We developed a computational pipeline to design guide RNAs for targeted Vg knockout, evaluating candidate sites in functional exons based on RNA secondary structure thermodynamics and frameshift potential. Comparative analysis revealed complementary strengths in two lead candidates. The exon 2 target site exhibits markedly weaker secondary structure (ΔG = − 0.25 kcal/mol versus − 2.10 kcal/mol for exon 3), aligning with empirical evidence that sites with ΔG > − 1.0 kcal/mol achieve 2–5× higher Cas9 binding efficiency. This site yielded moderate frameshift frequency (77.8%; 61.9 percentile). Conversely, the exon 3 target, despite stronger structural constraints, demonstrated superior functional disruption metrics demonstrating very high frameshift frequency (88.3%; 95.2 percentile), high editing precision, minimal microhomology-mediated repair bias, and reproducible outcomes wherein nearly all predicted indels disrupt the coding sequence. We recommend parallel empirical validation of both exon 2 and exon 3 targets to resolve the trade-off between structural accessibility (favoring higher editing rates) and frameshift efficacy (favoring complete loss-of-function). This dual-target strategy accommodates uncertainty in in vivo performance while maximizing the probability of generating informative phenotypes. Our in silico framework enables rational CRISPR design in non-model organisms by computationally balancing biophysical accessibility with functional impact, accelerating functional genomics in species where empirical optimization faces substantial biological constraints.

Abstract One-pot CRISPR diagnostics face a fundamental incompatibility: nucleic acid amplification requires rapid target accumulation, whereas CRISPR activation irreversibly consumes those substrates, destabilizing reaction kinetics. Existing strategies rely on empirical parameter balancing or external staging but lack an intrinsic mechanism to enforce reaction order within a single reactor. Here we introduce thermodynamic encoding as a molecular design principle that programs reaction order directly into DNA primers, enabling autonomous, threshold-gated activation of CRISPR only after sufficient amplicon accumulated. By embedding a defined free-energy differential between competing primers, the system evolves through two kinetically ordered amplification regimes, decoupling amplification from CRISPR signal transduction without physical separation or external triggers. This architecture relocates PAM dependence from native genomic targets to primer-encoded design, enabling detection of otherwise inaccessible loci while preserving single-nucleotide discrimination. An ordinary differential equation model captures the threshold behavior and establishes a predictable framework for primer design. Building on this principle, we develop Thermodynamically Encoded Molecular Programming for One-pot diagnostics (TEMPO), which achieves attomolar sensitivity within 30 min and enables sequencing-concordant SNP genotyping and pathogen detection in a single-step microfluidic format.

Abstract The mitochondrial phosphatase PPTC7 has emerged as a potent regulator of metabolism and mitophagy as its global knockout leads to perinatal lethality in mice. However, no known Mendelian diseases have been linked to PPTC7 deficiency, rendering its role in human pathophysiology unclear. Here, we identify two independent homozygous variants in PPTC7 : a missense variant, p.D158N, and a duplication variant (c.*57dup) within the 3` untranslated region (UTR). These variants were detected in three patients from two unrelated families presenting with a primary mitochondrial disease characterized by hypomyelinating leukodystrophy, recurrent metabolic and lactic acidosis, and anemia with immune dysregulation. Patient samples, including plasma and primary fibroblasts, showed robust metabolic and mitochondrial dysfunction, with substantial phenotypic overlap with Pptc7 knockout murine fibroblast models. PPTC7 patient fibroblasts carrying the p.D158N variant and CRISPR-knocked in cells to model the 3`UTR variant showed hallmarks of excessive BNIP3- and NIX-mediated mitophagy, including aberrant mitochondrial morphology, diminished mitochondrial protein expression, and increased mt-Keima flux. Critically, increased mitophagy in these cellular models was rescued by exogenous PPTC7 expression, confirming dysfunction derives from loss of this mitochondrial phosphatase. Mechanistically, we found that the p.D158N variant, affecting a highly conserved residue, disrupts metal binding to compromise both the enzymatic phosphatase function of PPTC7 as well as its negative regulation of BNIP3 and NIX. Collectively, these data provide the first known cases with a recessive inborn error of mitophagy due to PPTC7 deficiency and underscore the importance of this mitochondrial phosphatase in maintaining metabolic health and balanced mitophagy.

Abstract The research was focused on extracting Bacillus spp. and genomic-wide analysis from locally fermented yogurt to assess their potential as probiotics through genetic characterization. Two bacterial strains, JF-5 and isolate JY-2 were isolated from total 25 yogurt samples. The samples were collected from markets in Rawalpindi and Islamabad. Isolation was carried out using BHI agar as culture medium. The tested isolates received morphological and biochemical examinations, biosafety testing and enzymatic evaluations to evaluate their preliminary probiotic-associated traits. The probiotic characteristics of both strains differed, although they shared Gram-positive morphology, together with rod-shaped features and exhibited catalase activity. Analyses demonstrated that isolate JF-5 possessed proteolytic and lipolytic capacities and showed resilience against bile and pH variations. However, isolate JY-2 displayed DNase activity in addition to hemolysis, making it unsuitable for use as a safe probiotic. Whole-genome sequencing was applied to the strain JF-5 as the next analysis step. The bacterial strain was defined as Bacillus altitudinis through genome sequencing, which showed a 3.77 Mbp genome size alongside 41.2% GC content and 3962 coding sequences. The antiSMASH analysis platform detected various biosynthetic gene clusters that can produce antibacterial and probiotic traits, such as lichenysin, bacilysin, fengycin and siderophore compounds. CRISPR -Cas systems and vancomycin resistance-related genes (vanG, vanY, vanT) were identified with analysis. These antimicrobial resistance determinants have important biosafety implications especially with regard to possible horizontal gene transfer. This is why the strain cannot be proposed to be implemented as probiotics without a careful study of its safety. Even though the genomic presence indicates environmental adaptability through the presence of stress response genes, sporulation genes, and nutrient assimilation genes, alone in the genome does not indicate functional probiotic efficacy. This investigation presents the first detailed genomic analysis of indigenous Pakistan-based probiotic Bacillus strains. Whole-genome sequencing were performed by MicrobesNG Lab, Birmingham, United Kingdom.