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 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 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.

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.

Abstract Cyanobacteria are important primary producers and are used in biotechnology as microbial cell factories due to their ability to use solar light for oxygenic photosynthesis. Synechocystis sp. PCC 6803 is a popular model cyanobacterium, yet there are ambiguities in the precise coding regions of many genes, and numerous genes encoding small proteins have remained undetected. Here we present the results of a ribosome profiling (Ribo-seq) analysis involving inhibitors that stall ribosomes at translation initiation and termination sites (TIS- and TTS-Ribo-seq), combined with a proteogenomic reevaluation and reannotation of its entire genome. We report evidence for the translation of 3,050 annotated genes based on proteogenomics (83%), of 3,492 based on Ribo-seq (95.2%), and of 3,009 supported by both methods (82%). The data suggested both novel protein-coding genes and corrections for annotated ones. We validated 15 novel small proteins translated from antisense RNAs, from intergenic and intragenic regions and identified 69 novel, mostly small proteins based on proteogenomics. With slr0489, slr1079 and slr1082 we identified three genes with ~300 nt long intragenic out-of-frame coding regions and show that both the internal and host reading frames are translated. The resulting proteins interact with each other, resembling certain defense or toxin-antitoxin systems. Our data illustrate the enormous value of consolidating genome annotations in the context of integrated experimental data and suggest that genome annotations in general need to be extended and revised. All of our data can be accessed via an intuitive and interactive genome browser platform at https://www.bioinf.uni-freiburg.de/~ribobase/.

Chimeric antigen receptor (CAR)-T cell therapy has transformed the management of hematologic malignancies but faces obstacles, including severe treatment-related toxicities, highly suppressive tumor microenvironment (TME), inadequate long-term persistence, and poor trafficking/infiltration into solid tumor. This review summarizes recent genetic engineering strategies designed to overcome these barriers and to improve the safety, durability, and spatial effectiveness of CAR-T cell therapy. To mitigate cytokine release syndrome and neurotoxicity, approaches such as affinity-tuned and humanized scFvs, hinge/TM optimization, ITAM calibration have been developed alongside programmable “switch-off” and “switch-on” systems incorporating suicide genes, antibody-bridging switches, and optogenetic or hypoxia-gated circuits. TME remodelling strategies leverage nanomaterials for localized cytokine delivery, cell-surface “backpack” systems, and oncolytic viruses engineered to release cytokines or checkpoint-blocking biologics. Enhancing durability and resistance to exhaustion increasingly relies on precision genome engineering, including CRISPR-based editing and multiplexed shRNA platforms targeting inhibitory receptors and exhaustion-driving transcriptional programs. Finally, chemokine-receptor engineering and local biomaterial-based delivery systems are discussed as routes to improve CAR-T trafficking and intratumoral persistence. We also highlight the remaining translational challenges including checkpoint redundancy, in vivo payload dilution, vector capacity limits, and the safety of multiplex genome editing. Collectively, these interdisciplinary innovations point towards integrated, patient-tailored CAR-T platforms that combine safety control, metabolic and transcriptional resilience, and improved TME navigation to enable broader clinical application.

Epilepsy affects more than 50 million individuals worldwide, and approximately one-third of patients remain refractory to existing antiseizure medications. Advances in gene therapy and genome editing have opened new possibilities for disease-modifying interventions that directly target the molecular and circuit-level mechanisms underlying epileptogenesis. Recent progress in central nervous system tropic viral vectors, non-viral delivery systems, and programmable genome-editing technologies has enabled precise manipulation of neuronal and glial function in preclinical epilepsy models. Strategies range from restoration of haploinsufficient genes implicated in monogenic epilepsies, such as SCN1A in Dravet syndrome, to modulation of neuronal excitability through engineered ion channels, neuropeptides, and astrocyte-based approaches. In parallel, CRISPR-derived platforms, including transcriptional activation and repression systems, base editing, and prime editing, offer new avenues for regulating gene expression in post-mitotic neurons without introducing double-strand DNA breaks. Despite these advances, significant translational challenges remain, including efficient and cell-type-specific delivery, long-term safety, and the risk of network-level side effects in the epileptic brain. This review critically examines recent gene therapy and genome-editing approaches for epilepsy, highlights key technological and biological barriers to clinical translation, and discusses emerging strategies that may enable durable and targeted treatments for drug-resistant epilepsies.

Abstract Tagging a gene endogenously can identify when the gene is expressed and where the protein is localized. CRISPR is the primary tool for generating tags of endogenous genes, but it is error-prone and requires unique reagents for each gene and tag. Recombinases can insert DNA in an error-free and modular manner. Here, we tested eight recombinases for germline function in the nematode C. elegans, and introduce PhIT, a recombinase-based method for protein tagging. First, a short 39bp PhiC31 attB landing pad is inserted into the locus by CRISPR. This strain is a resource which can be used to insert a variety of modular tags. Second, tags are inserted by the integrase PhiC31, and in tandem, extraneous backbone sequences are removed by a tyrosine recombinase. Current modular tags include seven different fluorescent proteins, FLP-regulated cell-specific expression constructs, and degron tags. Importantly, tags can be inserted by genetic crosses instead of by microinjection.

Abstract Bacillus subtilis is a pivotal model organism in both industrial biotechnology and scientific research, where the efficiency of its genetic engineering is very important. However, achieving highly efficient gene insertion in this bacterium remains a significant technical challenge. To address this, we aimed to develop a novel gene insertion tool in B. subtilis . Building upon the Vibrio cholerae -derived Vch CAST system, we systematically optimized and successfully established a high-performance VchCAST system. The core components of this system include the TniQ-Cas678 complex, a guide RNA for precise targeting, and the TnsABC transposase complex responsible for DNA integration. Under antibiotic selection, screening and employing a strong promoter to drive crRNA expression increased the single-locus transposition efficiency to 41%. Subsequent genomic integration of the transposase operational unit further enhanced the efficiency to 80%. Moreover, we demonstrated that overexpressing the auxiliary factor BmrR enables simultaneous integration at two distinct genomic loci. Through protein engineering of the key transposase TnsB, we obtained optimized variants V178F and V178L with significantly enhanced activity, which improved the overall transposition efficiency by 232.6% and 178.07%, respectively. We then conducted transposition validation with the optimized system, achieving a site-specific gene insertion efficiency of approximately 95.25%. In conclusion, this study not only provides a robust gene insertion platform for B. subtilis microbial cell factory engineering, but also stands as a valuable reference for the construction of gene insertion tool in other microbial.

Abstract CRISPR-Cas12a (Cpf1) offers distinct advantages for genome editing due to its flexible, T-rich PAM recognition. However, variable cleavage efficiency—modulated by sequence context and epigenetic features—remains a challenge, with existing predictors limited in accuracy and interpretability. Here, we present DeepCas12a, a hybrid deep learning framework integrating Convolutional Neural Networks (CNNs) and a Vision Transformer (ViT) encoder to capture both local sequence motifs and long-range dependencies. The model fuses DNA sequence data with epigenetic profiles (DNA methylation and chromatin accessibility) in an end-to-end architecture. Benchmarked on an independent test set, DeepCas12a outperformed state-of-the-art predictors, achieving an Average Precision of 0.783, an AUC of 0.868, and a Spearman correlation of 0.630. Furthermore, interpretability analysis via saliency maps confirms the model captures biologically relevant features, including PAM specificity and seed region sensitivity, facilitating rational guide RNA design.

Abstract The U6 promoter plays a pivotal role in the CRISPR/Cas9 system by driving the transcription of single guide RNA (sgRNA), which directs Cas9 to achieve precise genome editing. Endogenous U6 promoters typically exhibit superior transcriptional activation efficiency compared to exogenous counterparts, thereby enhancing the efficacy of genome editing. However, the endogenous U6 promoter in kenaf ( Hibiscus cannabinus L.) remains uncharacterized. In this study, we conducted a homologous search of the kenaf genome using the Arabidopsis U6 (AtU6-26) RNA sequence as a reference, identifying two candidate promoters, HcU6-1 and HcU6-14. Promoter fragments were amplified from the genomic DNA of kenaf cultivar 'Fuhong 952' and subsequently cloned into a GUS fusion expression vector. Histochemical staining revealed transcriptional activity for both promoters, with HcU6-14 demonstrating significantly stronger activity. To evaluate editing efficiency, we constructed a CRISPR/Cas9 vector containing HcALS sgRNA, driven by either the kenaf U6-14P promoter or the cotton U6-9P (GbU6-9P) promoter. Kenaf hairy roots were regenerated via Agrobacterium rhizogenes K599-mediated transformation. Sequencing analysis of ALS gene fragments from these hairy roots confirmed successful targeted editing when using the kenaf U6-14P promoter, whereas no base mutations were detected with the cotton U6 promoter. These findings highlight the superior editing efficiency of the kenaf U6 promoter and provide a critical foundation for advancing functional genomics research in kenaf.

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related death, yet adequate in vitro models mimicking the tumor immune microenvironment (TIME) are rare. Specifically, the role of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in modulating interactions between tumor cells and tumor-associated macrophages (TAMs) is not fully understood. We established a 3D multicellular tumor spheroid (MCT) model using murine N-HCC25 cells with CRISPR/Cas9-mediated knockouts of Nrf2 and its negative regulator Kelch-like ECH-associated protein 1 (Keap1), the latter mimicking constitutive activation. N-HCC25 cells were co-cultured with bone marrow-derived macrophages (BMDMs) isolated from wild-type and Nrf2-knockout C57BL/6J mice. We compared co-culture setups (conditioned media, transwell systems, direct contact) using RT-qPCR, flow cytometry, and invasion assays. 3D spheroid systems better preserved stemness than 2D cultures and revealed functional Nrf2-dependent effects such as increased Vegf-α secretion in Keap1-deficient spheroids. Among the different co-cultivation models, most profound effects were observed in the MCT model. Macrophages successfully integrated into the spheroids and triggered invasive outgrowth, whereas MCTs containing Nrf2-deficient macrophages displayed markedly reduced invasion and lower programmed cell death ligand-1 expression. These findings demonstrate that Nrf2 signaling in macrophages fosters an immunosuppressive and pro-invasive microenvironment. The established MCT model provides a suitable platform to further unravel Nrf2-dependent mechanisms in the HCC TIME.

Abstract Apoptosis is a highly conserved form of programmed cell death controlled by a core molecular pathway that was first defined in Caenorhabditis elegans and is conserved in mammals. This pathway is composed of egl-1/ BH3-only, ced-9 /Bcl-2, ced-4 /Apaf-1, and ced-3/ Caspase. Despite being discovered more than 20 years ago, tissue-specific apoptosis induction as well as endogenous expression pattern and dynamic subcellular localization of apoptosis proteins remain incompletely defined. Here, we generated a complete set of CRISPR/Cas9-engineered transcriptional and translational reporters for all four apoptosis genes and systematically analyzed their expression and subcellular localization in the C. elegans germline and embryo. We show that somatic apoptosis is driven by precise, lineage-specific activation of egl-1 , whereas ced-9 , ced-4 , and ced-3 are ubiquitously expressed. In contrast, DNA-damage triggers a robust CEP-1/p53-dependent-induction of egl-1 throughout the germline, yet apoptosis occurs only in late pachytene cells. We also identify intron1 of egl-1 as essential for CEP-1–dependent transcriptional activation. Analysis of brc-1 and syp-2 mutants demonstrates that distinct meiotic surveillance pathways converge on egl-1 induction. Analysis of the subcellular localization of the downstream regulators CED-9, CED-4, and CED-3 reveals dynamic, tissue-specific localizations that refine the classical apoptosis model. CED-4 transitions from a perinuclear distribution in the germline and early embryos to a predominantly mitochondrial localization later in embryogenesis, while CED-3 changes its subcellular localization depending on developmental stage and apoptotic status. CED-9 localizes to distinct mitochondrial foci in both embryo and germline. Together, these reporters reveal that C. elegans apoptosis is governed by two mechanistically distinct programs: (1) lineage-specific egl-1 activation in embryos and (2) checkpoint-mediated activation of egl-1 in the germline, where additional, yet unidentified pathways restrict apoptotic execution. These reporters also provide a comprehensive toolbox for dissecting apoptotic and non-apoptotic functions of the conserved apoptotic machinery in vivo .

The integration of 3D bioprinting technology and CRISPR-Cas9 genome editing has become a game-changing method for creating complex organotypic cancer models. This integrated platform overcomes the drawbacks of traditional 2D culture systems by enabling precise genetic modifications within physiologically relevant, biomimetic tumor microenvironments. Researchers can more precisely recreate tumor progression, oncogenic mutations, cellular heterogeneity, and drug resistance mechanisms by utilizing the structural complexity provided by 3D bioprinting and the specificity of CRISPR-Cas9-mediated gene editing. CRISPR-Cas9 enables specific gene modifications, including oncogene knockout (e.g., MYC, KRAS) or immune checkpoint genes (e.g., PD-1, PD-L1), in 3D-bioprinted structures made from tumorigenic or patient-specific cell populations. It has been demonstrated that these modified models maintain important histopathological and molecular characteristics of original tumors, allowing for accurate high-throughput screening of immunotherapeutics and anticancer drugs. Significantly speeding up the modeling of tumorigenesis, studies using prostate cancer organoids showed gene correction efficiencies ranging from 50 to 90 %. Additionally, in 3D cultures, combinatorial CRISPR-Cas9 editing has demonstrated synergistic drug responses in models of lung and breast cancer, underscoring the platform's potential for discovering new therapeutic targets. Biomaterial-based vectors, like hydrogels and nanocarriers, are being improved to reduce off-target effects and increase intracellular uptake to increase the accuracy and safety of CRISPR delivery. However, issues with scalability, reproducibility, and standardization still exist, requiring ongoing interdisciplinary cooperation to improve downstream validation procedures, gene-editing tactics, and bioink formulations. The potential of CRISPR-Cas9-integrated 3D bioprinting as a state-of-the-art technique for drug discovery and cancer modeling is highlighted in this review. It emphasizes how the platform can speed up translational research in oncology, lessen dependency on animal models, and customize treatment plans. The goal of this review is to present a thorough summary of current developments, technical difficulties, and potential paths forward in this quickly developing field.

Abstract Background To investigate the distribution of CRISPR-Cas systems in Escherichia coli ( E. coli ) isolates and evaluate their associations with multilocus sequence types (MLST), antimicrobial resistance genes (ARGs), and plasmid features. Results ST1193 (16/65, 24.6%) and ST95 (11/65, 16.9%) were the predominant lineages. ST1193 showed a higher resistance gene burden than ST95, and bla TEM−1 was detected in 87.5% of ST1193 isolates. CRISPR-Cas systems were detected in 22 isolates (33.8%), including 11 with type I-F (50.0%), 10 with type I-E (45.5%), and one with both types. Spacer sequences were primarily directed against plasmid DNA. Plasmid replicons were frequently detected, and plasmid burden varied across lineages. All the ST1193 isolates lacked detectable CRISPR-Cas systems, whereas 90.9% (10/11) of ST95 isolates harbored type I-F systems. Conclusions CRISPR-Cas carriage was strongly lineage-dependent and showed an inverse association with predicted resistance gene burden in this cohort; this pattern should be interpreted as a lineage-structured correlation rather than mechanistic evidence of CRISPR-mediated restriction of ARG acquisition.

Abstract A growing body of evidence supports that targeting the tumour extracellular matrix (ECM) in solid cancers holds great promises to reactivate T cell migration in immune-excluded patients. By matrisome profiling of triple-negative breast cancer (TNBC) patients, we identified two core ECM proteins enriched in fibrotic immune-excluded tumours and associated with reduced CD8+ T cell stromal infiltration, versican (VCAN) and fibronectin (FN1). Both cancer-associated fibroblasts and aggressive cancer cell lines were found to deposit these two proteins in vitro, conferring resistance to T-cell-mediating cytotoxicity. Characterisation of in vivo murine breast cancer models 4T1 and EMT6 revealed significant differences in tumour ECM deposition and immune cell composition. Accordingly, targeting Fn1 and Vcan in both models induced opposite effects on tumour growth. While it appeared unfavourable in inflamed non fibrotic tumours, deletion of Fn1 in cancer cells was beneficial in immune-excluded tumours by promoting TCF7+ T cells and restoring anti-PD1 response.