Search Results (662)

Primary hepatocytes are limited by poor proliferative capacity and a finite replicative lifespan, restricting their utility in long-term in vitro studies. Here, we report the generation of expandable hepatocyte-like progenitor cells from GGTA1 knockout pigs, a large-animal model with reduced immunogenicity. Porcine fibroblasts were directly reprogrammed using a non-integrative episomal system encoding hepatic transcription factors, enabling stable lineage conversion without genomic integration. A simplified two-vector configuration combined with codon optimization enabled evaluation of vector-dependent effects while maintaining genomic safety without viral integration. The resulting cells exhibited hepatocyte-like morphology and gene expression, and transcriptomic analysis revealed a progressive shift toward liver-associated profiles during extended culture. Chromosomal analysis revealed vector-dependent differences in genomic stability, with codon-optimized cells showing increased aneuploidy, indicating a trade-off between proliferative capacity and genomic integrity. The cells also demonstrated sustained proliferative capacity, supported by maintenance of telomere length, increased expression of TERT and MYC, and reduced CDKN1A levels. Importantly, sustained proliferation was supported by complementary evidence from chromosomal and telomeric analyses. Although chromosomal alterations were observed during long-term culture, their biological significance remains to be fully determined. These cells partially recapitulate hepatocyte functions and provide a renewable in vitro system for studies of hepatic biology, proliferation, drug metabolism, toxicity, and repeated in vitro applications. Full article

Background/Objectives: Immune checkpoints are critical regulatory pathways that maintain peripheral tolerance and prevent autoimmunity. Among these, the programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) axis serves as a major inhibitory pathway that terminates T cell responses. While protein-based checkpoint blockade (ICB) targeting this axis has revolutionized clinical cancer therapy, its clinical efficacy is frequently limited by low response rates, immune-related adverse events (irAEs), and the emergence of adaptive resistance. To break through these bottlenecks, genetic interruption has emerged as a high-precision alternative to modulate the PD-1/PD-L1 pathway at the nucleotide level. Methods: A comprehensive systematic review of literature was performed across major databases (PubMed, Web of Science), with a focus on high quality studies published up to 2026. Results: Direct genomic disruption via CRISPR/Cas9 and post-transcriptional silencing through RNA interference can effectively neutralize inhibitory signaling at its source. Recent advances demonstrate that targeting upstream regulatory nodes—including metabolic checkpoints (e.g., lactate metabolism) and biophysical mechanisms (e.g., liquid–liquid phase separation)—provides superior transcriptional control over PD-L1. Furthermore, engineering CAR-T cells with multiplex gene editing (e.g., TCR/B2M/PD-1 knockout) or localized scFv secretion significantly enhances antitumor potency while reducing systemic toxicity. Innovations in organ-targeted lipid nanoparticles and stimuli-responsive biomimetic carriers further address the delivery barriers in solid tumors. Conclusions: Gene therapy provides a high-precision platform for PD-1/PD-L1 modulation, offering a viable strategy to overcome adaptive resistance. Future clinical application depends on the refinement of safer editing tools, such as base editing, and the standardization of intelligent delivery systems to ensure controllable and scalable cancer immunotherapy. Full article

Background/Objectives: Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer. Human ccRCCs have increased glycolytic metabolism and decreased mitochondrial oxidative metabolism relative to normal kidneys. Our research using human RCC4 ccRCC cells and a murine model of ccRCC, TRACK (TRAnsgenic model/Cancer/Kidney), in which a triple-mutant (P402A, P564A, N803A) human HIF1α is selectively expressed in proximal tubule cells (PTCs), revealed highly induced ATF4, a stress-responsive transcription factor. We then investigated the role of ATF4 in the metabolic changes in ccRCC. Methods: We performed comprehensive analysis of the ccRCC Cancer Genomics Atlas (TCGA) data. We deleted ATF4 in PTCs of TRACK mice and human RCC4 cells. We conducted genome-wide transcriptomic and untargeted metabolomic studies of cortices of WT and CGERA∆T (TRACK mice with PTC-specific ATF4-knockout (KO)) mice and performed glucose isotopologue tracing in parental and ATF4 KO RCC4 cells. Results: Analysis of TCGA data showed increased mRNAs of enzymes in glycolysis and reduced mRNAs of enzymes in the TCA cycle. Transcriptomic and metabolomic studies demonstrated that ATF4 deletion suppressed glycolysis and enhanced TCA cycle metabolism in CGERA∆T versus WT cortices. Glucose isotopologue tracing showed that ATF4 deletion altered glycolysis pathway metabolite levels and shifted glucose metabolism towards the TCA cycle, evidenced by increased intracellular [¹³C₂]citrate in RCC4-ATF4 KO cells. Using the Seahorse XFe96 analyzer we also showed reduced glycolytic capacity and reserve in RCC4-ATF4 KO cells. Conclusions: Collectively, our results demonstrate that ATF4 regulates glycolysis in ccRCC, supporting ATF4 as a therapeutic target. Full article

Rapeseed (Brassica napus L.) growth and productivity are severely constrained by drought stress worldwide. Stomata are central regulators of plant transpiration and gas exchange, and therefore, represent key targets for enhancing water-use efficiency and drought tolerance. The transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4), a key regulator of the abscisic acid (ABA) signaling pathway, plays crucial roles in plant abiotic stress responses and stomatal regulation. Nevertheless, the biological functions of BnaABI4 in B. napus remain largely unclear. In this study, four BnaABI4 paralogs were identified in the elite rapeseed cultivar ZS11 through genome-wide identification and comprehensive bioinformatic analyses. Each BnaABI4 protein harbors only one conserved AP2 domain, and their promoters contain multiple stress/hormone-responsive cis-regulatory elements (CREs). We subsequently generated BnaABI4-4 overexpression (OE) lines as well as BnaABI4 CRISPR/Cas9-mediated knockout (KO) transgenic lines. Phenotypic assays demonstrated that OE line had reduced transpiration rate (Tr), stomatal conductance (Gs) and stomatal density, along with enhanced drought tolerance, whereas KO lines showed the opposite phenotype. Transcriptome profiling identified numerous differentially expressed genes (DEGs) enriched in biological pathways associated with stomatal regulation, ABA signal transduction, and drought acclimation. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses confirmed significant enrichment of DEGs in processes including stomatal development, stomatal movement, reactive oxygen species (ROS) homeostasis, and drought tolerance. Collectively, our findings demonstrate that BnaABI4 negatively regulates stomatal density while positively contributing to drought tolerance in B. napus. This study lays a mechanistic foundation for genetic improvement and molecular breeding of drought-tolerant rapeseed cultivars. Full article

Endothelial dysfunction is a key characteristic of many diseases, including atherosclerosis, hypertension, heart failure, stroke, cancer, acute respiratory distress syndrome (ARDS), peripheral vascular disease, coronavirus 2019 (COVID-19), and pulmonary arterial hypertension (PAH). To improve understanding of the roles of endothelial cells (ECs) in health and disease, EC-specific genome editing technologies have been developed in recent years. Therapeutic strategies that aim to restore a healthy endothelial monolayer include the inhibition of endothelial genes that cause EC injury and dysfunction and the induction or activation of endothelial genes that drive EC repair and regeneration. In this review, we describe established recombinase-mediated genetic modification technologies and emerging EC-specific genome editing technologies including viral and non-viral delivery of the CRISPR/Cas9 genome editing system, and we summarize the strengths and limitations of each technology. We then discuss possible avenues for future research, including the development of organ-specific EC genome editing technologies. In short, EC-specific genome editing technologies can be used to modulate gene expression selectively in ECs and even within a specific vascular bed and/or distinctive EC subtype, and, in doing so, greatly improve the understanding of vascular biology and help develop precision genetic medicine targeting the disease-causing vascular bed(s) to effectively treat diseases caused by vascular endothelial dysfunction. Full article

Plant diseases remain a major constraint to crop productivity and global food security. Conventional breeding has long been used to develop resistant cultivars through the introgression of resistance traits from wild relatives and the selection of favorable phenotypes. However, this process is often slow and limited by linkage drag, known genetic diversity, intrinsic genetic limitations, and the rapid evolution of pathogen populations. Molecular breeding strategies, including marker-assisted selection and genomic selection, have improved the precision of resistance breeding but still rely on existing genetic variation. Recent advances in genome editing technologies are transforming plant breeding by enabling precise modification of gene targets. CRISPR-based systems allow targeted gene knockouts, promoter editing, allelic replacement, and multiplex editing to rapidly generate resistance traits. Many studies have demonstrated that editing susceptibility genes or regulatory regions can enhance resistance to diverse pathogens. Recent research shows that resistance can also be improved by targeting non-classical genes involved in plant immunity, including transcription factors, membrane transporters, heat shock proteins, cell wall-related genes, metabolic enzymes, and epigenetic regulators. Emerging tools such as base editing, prime editing, regulatory tools, and transposon-associated genome engineering systems are further expanding the precision and versatility of plant genome editing. Despite these advances, challenges related to delivery systems, editing efficiency, regulatory frameworks, and field validation remain. Continued technological progress and improved knowledge of plant immune networks will be essential to fully integrate genome editing into crop improvement programs. Full article

Prostate cancer (PCa) is immunologically “cold” and resistant to immune checkpoint blockade (ICB), yet bulk analyses show low, non-prognostic PD-L1 expression. We hypothesised that this paradox reflects two overlooked dimensions: basal heterogeneity (static engine) and IFN-$\gamma$-driven adaptive resistance (adaptive engine). Using TCGA-PRAD data ($n=554$) to parameterise an agent-based model, we simulated clonal selection and extended it to a hybrid discrete-continuum framework with reaction-diffusion IFN-$\gamma$. Bulk PD-L1 was low (median 1.48 TPM) and non-prognostic (HR $=1.15$, $p=0.621$). The static engine alone produced weak immunoediting (1.10-fold enrichment), whereas the adaptive engine drove a 2.95-fold enrichment of PD-L1-high clones via protective sanctuary formation, without increasing final tumour burden. Induction knockout ($P_{max}=0$) abrogated this advantage, while diffusion knockout ($D=0$) had no effect. The cold tumour paradox is resolved by a hierarchical twin engine: rare genomic outliers permit initial persistence, but local IFN-$\gamma$/PD-L1 feedback dominates resistance, identifying induction capacity as the primary therapeutic target for JAK/STAT inhibition combined with ICB. Full article

Background/Objectives: Acute myeloid leukemia (AML) remains in need of more broadly effective therapeutic strategies. THZ1, a covalent CDK7 inhibitor, has shown anti-leukemic activity in AML, but the mechanism underlying its response remains incompletely defined. This study aimed to identify key modulators and related transcriptional programs involved in THZ1 response in AML. Methods: We combined a genome-wide CRISPR/Cas9 screen with functional validation and transcriptomic analyses. Results: TP53 was the top positively selected gene in the CRISPR screen in MOLM-13 cells. THZ1 induced p53 accumulation, and pharmacologic activation of the p53 pathway with idasanutlin further enhanced the early anti-leukemic response to THZ1 at 24 h. TP53 loss reduced the sensitivity of AML cells to THZ1-induced apoptosis, but did not abolish THZ1 responsiveness. Transcriptomic analyses showed that TP53 loss substantially reshaped the transcriptional state, whereas MYC and E2F target programs remained the most consistent pathways linked to THZ1 response. THZ1 also continued to suppress these programs in TP53-knockout MOLM-13 cells and TP53-mutant THP-1 cells. Conclusions: Overall, TP53 contributes to the anti-leukemic response to THZ1 but does not determine it, and THZ1 continues to suppress MYC and E2F target programs after TP53 loss. Full article

Induced resistance primes host immunity for enhanced protection; however, how pathogens respond to this primed state remains poorly understood. Here, we investigated the molecular responses of the rice blast fungus Magnaporthe oryzae during infection of benzothiadiazole (BTH)-primed rice. Seed priming with BTH conferred long-lasting resistance against M. oryzae at the four-leaf stage. Time-course transcriptomic analyses (12–48 hpi) identified 699 differentially expressed genes (DEGs) in M. oryzae, revealing a distinct temporal transition during infection of BTH-primed rice. The fungal transcriptional response shifted from early growth and environmental sensing to enhanced protein turnover, metabolic repression, energy depletion, and genomic instability, indicating progressive impairment of fungal fitness by host immunity. From these DEGs, eight BTH-suppressed candidate virulence genes (MoBVG1–8) were selected for functional characterization. Gene overexpression analyses showed that two genes, MoBVG2 and MoBVG6, significantly increased pathogenicity on BTH-primed rice, while knockout analyses confirmed that both are required for full pathogenicity on non-primed control plants. MoBVG2 encodes a reactive oxygen species (ROS)-scavenging effector, and MoBVG6 encodes an environmental sensor, highlighting the importance of ROS detoxification and environmental perception for successful host colonization. Functional analyses further revealed that MoBVG2 contribute to vegetative growth, while MoBVG6 is required for proper appressorium development. Together, these findings suggest that BTH-induced resistance restricts blast disease by impairing fungal metabolic fitness and suppressing key virulence genes, providing novel insights into the pathogen-side molecular mechanisms underlying chemically induced resistance in plants. Full article

Histone methyltransferases of the Trithorax-related (ATXR) family act as critical epigenetic regulators in plants. However, systematic characterization of this gene family remains limited in the economically important oilseed crop Brassica napus. In this study, we performed a pan-genomic analysis of the BnaATXR family genes using 11 genetically diverse rapeseed accessions and identified a total of 185 BnaATXR family members, among which BnaATXR5 was categorized as a dispensable gene. Pan-genomic and phylogenetic analyses grouped these genes into five distinct subfamilies and uncovered strong sequence conservation and pervasive purifying selection across the family. Whole-genome duplication (WGD) was identified as the major evolutionary force driving BnaATXR genes expansion. Cis-acting regulatory element analysis further revealed significant enrichment of stress- and phytohormone-responsive motifs in the promoter regions of BnaATXR genes. BnaATXR members exhibited divergent tissue expression profiles: subfamilies B and C displayed constitutive and broad expression across multiple tissues, whereas subfamilies A and E exhibited pronounced tissue-specific expression, with preferential enrichment in reproductive organs. Notably, CRISPR/Cas9-mediated knockout of BnaATXR6 led to delayed flowering time, shortened siliques, and decreased seed size, thereby demonstrating a key functional role of this gene in the modulation of yield-associated agronomic traits. Collectively, our findings present a genome-wide systematic characterization of the ATXR gene family and highlight their critical functional relevance to agronomically important traits in rapeseed. Full article

This paper systematically reviews the research progress on the physiological functions, gene cloning, classification basis, and expression regulation mechanisms of laccase in edible and medicinal fungi. Laccase is a copper-containing polyphenol oxidase widely distributed in these fungi, characterized by broad-spectrum substrate catalytic activity and redox properties. It plays a central role in lignin degradation, pigment synthesis, and environmental pollutant treatment. Regarding gene cloning, researchers have successfully isolated and identified laccase genes from multiple species using techniques such as transcriptome sequencing, RACE amplification, and gene knockout. Expression regulation studies have revealed that laccase genes exhibit stage-specific expression patterns during mycelial growth, fruiting body development, and lignin degradation. In recent years, breakthroughs in genomics, transcriptomics, and gene editing technologies have greatly advanced research into the cloning, classification, and regulatory mechanisms of laccase genes. This article systematically reviews the diversity, clonal classification, and regulatory mechanisms of these genes, aiming to provide a reference for further research and industrial development of laccase in edible and medicinal fungi. Full article

This study aimed to establish an efficient and precise cytosine base editor (CBE)-mediated knockout system in chicken somatic cells and primordial germ cells (PGCs). PGCs are pivotal for generating genome-edited chickens, but low transfection efficiency limit their application. Unlike CRISPR/Cas9, CBEs achieve precise C-to-T conversion without DNA double-strand breaks or donor templates, making them safer for avian genome engineering. We used CBEs to introduce premature stop codons in exon 1 of the sex-determining AR and DMRT1 genes for targeted knockout. Among 12 screened sgRNAs, sgRNA6 (AR, 94.67 ± 6.66%) and sgRNA9 (DMRT1, 6.67 ± 6.51%) performed best in DF-1 cells; in PGCs, their editing efficiencies reached 51.0% and 91.0%, respectively. No off-target mutations were detected in edited DF-1 cells. These findings confirm that CBE-mediated knockout is highly efficient and safe in chicken somatic and germ cells, providing a robust tool for avian genome editing. Full article

MicroRNAs (miRNAs) play important roles in the regulation of gene expression and are frequently dysregulated in cancer. Among them, the miR-196 family has been implicated in multiple malignancies, including colorectal cancer (CRC), but the isoform-specific transcriptional effects of miR-196A and miR-196B remain poorly understood. In this study, we generated miR-196A and miR-196B knockout SW48 CRC cell lines using CRISPR-based genome editing and performed RNA sequencing to investigate the transcriptional consequences of individual miR-196 isoform deletion. Transcriptomic analysis revealed widespread gene expression changes in both knockout models and demonstrated distinct clustering patterns between parental SW48 cells and miR-196-deficient cells. Functional enrichment analysis indicated that the altered genes were associated with biological processes related to cytoskeletal organization, intracellular transport, protein folding, and metabolic regulation. Notably, both shared and isoform-specific transcriptional alterations were observed, suggesting that miR-196A and miR-196B contribute to partially overlapping but distinct regulatory networks in CRC cells. Collectively, these findings provide a comprehensive transcriptomic overview of miR-196 isoform deletion in colorectal cancer cells and highlight potential isoform-dependent transcriptional programs that may contribute to CRC biology. Full article

Aflatoxin B1 (AFB1) is a prevalent and highly toxic mycotoxin in the food and feed chain and can directly injure the intestinal epithelium. Yet, its upstream determinants linking epithelial stress to cytotoxicity remain insufficiently defined. Here, we used porcine intestinal epithelial IPEC-J2 cells to characterize AFB1-induced cytotoxic and transcriptomic responses and to determine the role of the tight-junction scaffold, Cingulin-like 1 (CGNL1), a candidate gene identified through genome-scale CRISPR knockout library screening. The results showed that AFB1 exposure reduced cell viability in a dose-dependent manner and induced oxidative stress. RNA-seq profiling analysis revealed broad transcriptional remodeling, with activation of inflammatory pathways (including NF-κB and JAK–STAT signaling). Based on our constructed CGNL1-knockout IPEC-J2 cell line (CGNL1-KO IPEC-J2) using CRISPR/Cas9, it was found that CGNL1 deficiency markedly alleviated AFB1-induced cytotoxicity and oxidative stress. Comparative transcriptomics analysis showed that CGNL1 knockout attenuated AFB1-triggered aberrant expression of some CGNL1-dependent AFB1-responsive genes related to immune response under AFB1 challenge. Together, these findings identify CGNL1 as a potential modulator of epithelial susceptibility to AFB1 and support its involvement in the regulation of toxin-induced oxidative response. Full article

The proteolytic processing of the amyloid precursor protein (APP) is a core pathological event in Alzheimer’s disease (AD) pathogenesis, yet the global genetic regulatory networks modulating this process have not been fully characterized. To systematically identify novel regulators of APP cleavage, we performed a genome-wide CRISPR/Cas9 knockout screen utilizing an optimized UAS-GAL4-based cellular reporter, and identified genetic modulators governing amyloidogenic and non-amyloidogenic processing. The screen uncovered distinct functional gene clusters regulating the APP, prominently involving cellular metabolism, protein modification, and vesicular trafficking. Specifically, LDHB, PIAS2, CCDC53, and TRIM61 emerged as novel functional modulators. Biochemical validation confirmed that ablating these genes significantly alters the metabolic balance between sAPPα and amyloid-β (Aβ) production. Finally, integration with human AD transcriptomic datasets demonstrated that these identified modulators undergo significant dysregulation in clinics. Together, these findings establish a reporter-based functional screening framework for APP processing and identify candidate regulatory nodes linked to metabolism, protein modification, and vesicular trafficking. These candidates provide a resource for future mechanistic investigation and validation in more disease-relevant AD models. Full article