Search Results (315)

Conventional methods for generating knock-out or knock-in mammalian cell models using CRISPR-Cas9 genome editing often require tedious single-cell clone selection and expansion. In this study, we develop and optimise rapid and robust strategies to engineer homozygous fluorescent reporter knock-in cell pools with precise genome editing, circumventing clonal variability inherent to traditional approaches. To reduce false-positive cells associated with random integration, we optimise the design of donor DNA by removing the start codon of the fluorescent reporter and incorporating a self-cleaving T2A peptide system. Using fluorescence-assisted cell sorting (FACS), we efficiently identify and isolate the desired homozygous fluorescent knock-in clones, establishing stable cell pools that preserve parental cell line heterogeneity and faithfully reflect endogenous transcriptional regulation of the target gene. We evaluate the knock-in efficiency and rate of undesired random integration in the electroporation method with either a dual-plasmid system (sgRNA and donor DNA in two separate vectors) or a single-plasmid system (sgRNA and donor DNA combined in one vector). We further demonstrate that coupling our single-plasmid construct with an integrase-deficient lentivirus vector (IDLV) packaging system efficiently generates fluorescent knock-in reporter cell pools, offering flexibility between electroporation and lentivirus transduction methods. Notably, compared to the electroporation methods, the IDLV system significantly minimises random integration. Moreover, the resulting reporter cell lines are compatible with most of the available genome-wide sgRNA libraries, enabling unbiased CRISPR screens to identify key transcriptional regulators of a gene of interest. Overall, our methodologies provide a powerful genetic tool for rapid and robust generation of fluorescent reporter knock-in cell pools with precise genome editing by CRISPR-Cas9 for various research purposes. Full article

Viral oncolysis is considered a promising cancer treatment method because of its good tolerability and durable anti-tumor effects. Compared with other oncolytic viruses, Newcastle disease virus (NDV) has some distinct advantages. As an RNA virus, NDV does not recombine with the host genome, making it safer compared with DNA viruses and retroviruses; NDV can induce syncytium formation, allowing the virus to spread among cells without exposure to host neutralizing antibodies; and its genome adheres to the hexamer genetic code rule (genome length as a multiple of six nucleotides), ensuring accurate replication, low recombination rates, and high genetic stability. Although wild-type NDV has a killing effect on various tumor cells, its oncolytic effect and working mechanism are diverse, increasing the complexity of generating engineered oncolytic viruses with NDV. This study aims to employ whole-genome CRISPR-Cas9 knockout screening and RNA sequencing to identify putative key regulatory factors involved in the interaction between NDV and human colon cancer HCT116 cells and map their global interaction networks. The results suggests that NDV infection disrupts cellular homeostasis, thereby exerting oncolytic effects by inhibiting cell metabolism and proliferation. Meanwhile, the antiviral immune response triggered by NDV infection, along with the activation of anti-apoptotic signaling pathways, may be responsible for the limited oncolytic efficacy of NDV against HCT116 cells. These findings not only enhance our understanding of the oncolytic mechanism of NDV against colonic carcinoma but also provide potential strategies and targets for the development of NDV-based engineered oncolytic viruses. Full article

Alpha-Synuclein (α-Syn) is a presynaptic neuronal protein implicated in the pathogenesis of Parkinson’s disease (PD) and other synucleinopathies, primarily through its aggregation into insoluble fibrils. The extended α-Syn half-life necessitates treatment durations that are incompatible with efficient high-throughput drug screening, can risk compound stability or cause cellular toxicity. To address this, we inserted a PEST sequence, a motif known to promote rapid protein degradation, at the C-terminus of the SNCA gene using CRISPR/Cas9 to create a novel cell line with reduced α-Syn half-life. This modification accelerates α-Syn turnover, providing a robust model for studying α-Syn dynamics and offering a platform that is applicable to other long-lived proteins. Our results demonstrate a six-fold reduction in α-Syn half-life, enabling the rapid detection of changes in protein levels and facilitating the identification of molecules that modulate α-Syn production and degradation pathways. Using inhibitors of the proteasome, transcription, and translation further validated the model’s utility in examining various mechanisms that impact protein levels. This novel cell line represents a significant advancement for studying α-Syn dynamics and offers promising avenues to develop therapeutics for α-synucleinopathies. Future research should focus on validating this model in diverse experimental settings and exploring its potential in high-throughput screening applications. Full article

Prolonged expression of gene-editing components in CRISPR-modified plants can interfere with phenotypic analysis of target traits, increase the risk of off-target mutations, and lead to unnecessary metabolic burden. To mitigate these issues in peanut (Arachis hypogaea L.), callus-specific promoters were screened to restrict Cas9 expression to the callus stage, minimizing its activity in regenerated plants. In this study, six callus-specific genes in peanut were identified by mining RNA sequencing datasets and validating their expression profiles using quantitative reverse transcriptase PCR. The promoters of Arahy.H0FE8D, Arahy.WT3AEF, Arahy.I20Q6X, Arahy.ELJ55T, and Arahy.N9CMH4 were cloned and assessed for their expression activity. Beta-glucuronidase (GUS) histochemical staining confirmed that all five promoters were functional in peanut callus. Further investigation revealed their ability to drive cytosine base editing via a deaminase-nCas9 fusion protein, with all promoters successfully inducing precise base substitutions in peanut. Notably, P_Ah-H0FE8D, P_Ah-WT3AEF, P_Ah-ELJ55T, and P_Ah-N9CMH4 exhibited comparable or higher editing efficiencies than the commonly used cauliflower mosaic virus 35S promoter. These findings provide valuable tools for improving the biosafety of CRISPR-based genome editing in peanut breeding programs. Full article

Background/Objectives: The treatment of multiple myeloma (MM) remains a challenge, as almost all patients will eventually relapse. Proteasome inhibitors are a cornerstone in the management of MM. Unfortunately, validated biomarkers predicting drug response are largely missing. Therefore, we aimed to identify genes associated with drug resistance or sensitization to proteasome inhibitors. Methods: We performed genome-wide CRISPR-Cas9 knockout (KO) screens in human KMS-28-BM myeloma cells to identify genetic determinants associated with resistance or sensitization to proteasome inhibitors. Results: We show that KO of KLF13 and PSMC4 induces drug resistance, while NUDCD2, OSER1 and HERC1 KO cause drug sensitization. Subsequently, we focused on top sensitization hit, NUDCD2, which acts as a co-chaperone of Hsp90 to regulate the LIS1/dynein complex. RNA sequencing showed downregulation of genes involved in the ERAD pathway and in ER-associated ubiquitin-dependent protein catabolic processes in both untreated and carfilzomib-treated NUDCD2 KO cells, suggesting that NUDCD2 depletion alters protein degradation. Furthermore, bortezomib-treated NUDCD2 KO cells showed a decreased expression of genes that have a function in oxidative phosphorylation and the mitochondrial membrane, such as Carnitine Palmitoyltransferase 1A (CPT1A). CPT1A catalyzes the uptake of long chain fatty acids into mitochondria. Mitochondrial lipid metabolism has recently been reported as a possible therapeutic target for MM drug sensitivity. Conclusions: These results contribute to the search for therapeutic targets that can sensitize MM patients to proteasome inhibitors. Full article

Pediatric high-grade gliomas (pHGGs), particularly diffuse midline gliomas (DMGs), are among the most lethal brain tumors due to poor survival and resistance to therapies. DMGs possess a distinct genetic profile, primarily driven by hallmark mutations such as H3K27M, ACVR1, and PDGFRA mutations/amplifications and TP53 inactivation, all of which contribute to tumor biology and therapeutic resistance. Developing physiologically relevant preclinical models that replicate both tumor biology and the tumor microenvironment (TME) is critical for advancing effective treatments. This review highlights recent progress in in vitro, ex vivo, and in vivo models, including patient-derived brain organoids, genetically engineered mouse models (GEMMs), and region-specific midline organoids incorporating SHH, BMP, and FGF2/8/19 signaling to model pontine gliomas. Key genetic alterations can now be introduced using lipofectamine-mediated transfection, PiggyBac plasmid systems, and CRISPR-Cas9, allowing the precise study of tumor initiation, progression, and therapy resistance. These models enable the investigation of TME interactions, including immune responses, neuronal infiltration, and therapeutic vulnerabilities. Future advancements involve developing immune-competent organoids, integrating vascularized networks, and applying multi-omics platforms like single-cell RNA sequencing and spatial transcriptomics to dissect tumor heterogeneity and lineage-specific vulnerabilities. These innovative approaches aim to enhance drug screening, identify new therapeutic targets, and accelerate personalized treatments for pediatric gliomas. Full article

Seed development plays a critical role in determining both crop yield and grain quality in rice. As a key nutrient storage organ, the rice endosperm development not only contributes to grain filling but also plays an essential role during the early stages of seed germination. Amino acid metabolism is active during the process of seed development and seed germination. Asparagine is a primary amino acid responsible for long-distance organic nitrogen transport in plants. Asparagine synthetase catalyzes the synthesis of asparagine from aspartate and glutamine. In this study, CRISPR/Cas9-mediated knockout mutants of the OsASN2 gene of rice were generated. Homozygous mutants exhibited complete failure of seed germination, and heterozygotes could not produce homozygous offspring. Endosperm development of homozygous mutant seeds showed severe defects. Additionally, interacting protein screening combined with pull-down and co-immunoprecipitation (Co-IP) assays confirmed that OsASN2 physically interacted with pyruvate phosphate dikinase OsPPDKB, the mutants of which showed impaired endosperm development. These findings collectively indicate that OsASN2 plays a critical role in seed development and germination in rice. Full article

Hepatocellular carcinoma (HCC) presents significant intertumoral heterogeneity, complicating prognosis and treatment. To address this, we performed an integrated single-cell RNA-sequencing analysis of HCC specimens using Seurat and identified malignant cells via Infercnv. Through a systematic evaluation of 101 machine learning algorithms used in combination, we developed tumor-cell-specific gene signatures (TCSGs) that demonstrated strong predictive performance, with area under the curve (AUC) values ranging from 0.72 to 0.74 in independent validation cohorts. Risk stratification based on these signatures revealed distinct therapeutic vulnerabilities: high-risk patients showed increased sensitivity to sorafenib, while low-risk patients exhibited enhanced responses to immunotherapy and transarterial chemoembolization (TACE). Pharmacogenomic analysis with Oncopredict identified four chemotherapeutic agents, including sapitinib and dinaciclib, with risk-dependent efficacy patterns. Furthermore, CRISPR/Cas9-dependency screening prioritized SRSF7 as essential for HCC cell survival, a finding confirmed by the identification of protein-level overexpression in tumors via immunohistochemistry. This multi-omics framework bridges single-cell characterization to clinical decision-making, offering a clinically actionable prognostic system that can be used to optimize therapeutic selection in HCC management. Full article

Mpox is a zoonotic disease caused by the Mpox virus (MPXV). The rapid and accurate diagnosis of MPXV is essential for the timely and effective prevention, control, and treatment of the disease. In this study, we combined Multienzyme Isothermal Rapid Amplification (MIRA) (at 42 °C) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 12b(CRISPR/Cas12b) (at 60 °C) to develop a single-tube two-step assay for rapid MPXV detection, leveraging the distinct physical states of tricosane at these temperatures. MIRA amplification primers and CRISPR/cas12b SgRNA were designed based on the MPXV F3L gene. After screening the primers and sgRNAs, the reaction conditions were optimized, and the performances of the assay were evaluated. The detection limit (LOD) of this single-tube two-step MIRA-CRISPR/Cas12b assay for MPXV is four copies of DNA molecules. No cross-reactivity with other pathogens (herpes simplex virus (HSV), Epstein–Barr virus (EBV), Coxsackievirus A16 (CVA16), Enterovirus A71 (EV-A71), and measles virus (MeV)) was found. The assay also showed good consistency with quantitative real-time PCR (qPCR) (Kappa = 0.9547, p < 0.05, n = 100) in the detection of clinical samples, with a sensitivity of 98.5% and a specificity of 97.0%. The single-tube two-step MIRA-CRISPR/Cas12b assay permits the rapid (within 45 min), sensitive, and specific detection of MPXV. The lack of need for opening the reaction tube eliminates the risk of product contamination. Full article

Nucleic acid detection technology is crucial for molecular diagnosis. The advent of CRISPR/Cas12a-based nucleic acid detection has considerably broadened its scope, from the identification of infectious disease-causing microorganisms to the detection of disease-associated biomarkers. This innovative system capitalizes on the non-specific single-strand cleavage activity of Cas12a upon target DNA recognition. By employing a fluorescent probe in the form of a single-stranded DNA/RNA, this technology enables the observation of fluorescence changes resulting from nonspecific cleavage, thereby facilitating detection. CRISPR/Cas12a-based detection systems can be regarded as a new type of biosensor, offering a practical and efficient approach for nucleic acid analysis in various diagnostic settings. CRISPR/Cas12a-based biosensors outperform conventional nucleic acid detection methods in terms of portability, simplicity, speed, and efficiency. In this review, we elucidate the detection principle of CRISPR/Cas12a-based biosensors and their application in disease diagnostics and discuss recent innovations and technological challenges, aiming to provide insights for the research and further development of CRISPR/Cas12a-based biosensors in personalized medicine. Our findings show that although CRISPR/Cas12a-based biosensors have considerable potential for various applications and theoretical research, certain challenges remain. These include simplifying the reaction process, enhancing precision, broadening the scope of disease detection, and facilitating the translation of research findings into clinical practice. We anticipate that ongoing advancements in CRISPR/Cas12a-based biosensors will address these challenges. Full article

Congenital heart disease (CHD), the most common congenital anomaly, remains a significant lifelong burden despite advancements in medical and surgical interventions. Induced pluripotent stem cells (iPSCs) have emerged as a groundbreaking platform in CHD research, offering patient-specific models to investigate the genetic, epigenetic, and molecular mechanisms driving the disease. Utilizing technologies such as CRISPR/Cas9 gene editing, cardiac organoids, and high-throughput screening, iPSCs enable innovative strategies in disease modeling, precision drug discovery, and regenerative therapies. However, clinical translation faces challenges related to immaturity, differentiation variability, large-scale feasibility, and tumorigenicity. Addressing these barriers will require standardized protocols, bioengineering solutions, and interdisciplinary collaboration. This review examines the critical role of iPSCs in advancing CHD research and care, demonstrating their potential to revolutionize treatment through patient-specific, regenerative approaches. By addressing current limitations and advancing iPSC technology, the field is positioned to pave the way for precision-based CHD therapies for this lifelong condition. Full article

Foot and mouth disease virus (FMDV) is a highly pathogenic virus that mainly infects cloven hooved animals, such as pigs. The establishment of a rapid, sensitive and accurate point-of-care detection method is critical for the timely identification and elimination of infected pigs for controlling this disease. In this study, a RT-RAA-CRISPR/Cas13a method was developed for the detection of FMDV serotype O in pigs. Six pairs of RT-RAA primers were designed based on the conserved gene sequence of FMDV serotype O, and the optimal amplification primers and reaction temperatures were screened. The CRISPR-derived RNA (crRNA) was further designed based on the optimal target band sequence and the most efficient crRNA was screened. The results revealed that FMDV-O-F4/R4 was the optimal primer set, and the optimal temperature for the RT-RAA reaction was 37 °C. Moreover, crRNA4 exhibited the strongest detection signal among the six crRNAs. The established RT-RAA-CRISPR/Cas13a method demonstrated high specificity and no cross-reactivity with other common swine pathogens such as Senecavirus A (SVA), porcine reproductive and respiratory virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), and pseudorabies virus (PRV), additionally, it was observed to be highly sensitive, with a detection limit of 19.1 copies/µL. The repeatability of this method was also observed to be good. This method could produce stable fluorescence and exhibited good repeatability when three independent experiments yielded the same results. A validation test using three types of simulated clinical samples (including swab, tissue, and serum samples) revealed a 100% concordance rate. The detection results could be visualized via a fluorescence reader or lateral flow strips (LFSs). Thus, a highly specific and sensitive RT-RAA-CRISPR/Cas13a detection method was developed and is expected to be applied for the rapid detection of FMDV serotype O in situ. Full article

Pancreatic cancer is characterized by late diagnosis, therapy resistance, and poor prognosis, necessitating the exploration of early carcinogenesis and prevention methods. Preclinical mouse models have evolved from cell line-based to human tumor tissue- or organoid-derived xenografts, now to humanized mouse models and genetically engineered mouse models (GEMMs). GEMMs, primarily driven by oncogenic Kras mutations and tumor suppressor gene alterations, offer a realistic platform for investigating pancreatic cancer initiation, progression, and metastasis. The incorporation of inducible somatic mutations and CRISPR-Cas9 screening methods has expanded their utility. To better recapitulate tumor initiation triggered by inflammatory cues, common pancreatic risk factors are being integrated into model designs. This approach aims to decipher the role of environmental factors as secondary or parallel triggers of tumor initiation alongside oncogenic burdens. Emerging models exploring pancreatitis, obesity, diabetes, and other risk factors offer significant translational potential. This review describes current mouse models for studying pancreatic carcinogenesis, their combination with inflammatory factors, and their utility in evaluating pathogenesis, providing guidance for selecting the most suitable models for pancreatic cancer research. Full article

Background: Esophageal cancer (EC) is the eighth most common cancer and the sixth most common cause of death worldwide. Esophageal squamous cell carcinoma (ESCC) comprises the majority of esophageal cancers globally, and 5-Fluorouraci (5-FU) is one of the commonly used chemotherapeutics for this type of cancer. Chemoresistance to drugs is a main obstacle in the successful treatment of this malignancy. Methods: In this study, we used the CRISPR/Cas9 screening method to determine the target gene related to 5-FU drug resistance in esophageal cancer. Results: Our research findings indicate that the loss of PFKFB3 can increase the resistance of different human esophageal squamous cell carcinoma cell lines to 5-FU through various pathways. Specifically, in KYSE-70 cells, loss of PFKFB3 can induce epithelial–mesenchymal transition (EMT) and prolong the S phase of the cell cycle, allowing cancer cells to evade the effects of 5-FU and develop resistance. In the KYSE-270 and KYSE-150 cell lines, loss of PFKFB3 can upregulate the expression of Slug and Mcl-1, indirectly regulate Chk1 and promote its autophosphorylation, which in turn inhibits apoptosis, thus counteracting the effects of 5-FU. Conclusions: Our research not only enriches our understanding of the biological characteristics of different ESCC cell lines but also provides new clinical insights for future personalized treatments. Assessing the status of PFKFB3 can help predict resistance to 5-FU in ESCC patients with different genetic backgrounds, allowing for more precise treatment planning. This personalized approach has the potential to improve treatment efficacy, reduce unnecessary drug use and side effects, and ultimately improve patient survival rates and quality of life. Full article

Zebrafish (Danio rerio) has become a pivotal vertebrate model in biomedical research, renowned for its genetic similarity to humans, optical transparency, rapid embryonic development, and amenability to experimental manipulation. In recent years, the derivation of cell lines from zebrafish embryos has unlocked new possibilities for in vitro studies across developmental biology, toxicology, disease modeling, and genetic engineering. These embryo-derived cultures offer scalable, reproducible, and ethically favorable alternatives to in vivo approaches, enabling high-throughput screening and mechanistic exploration under defined conditions. This review provides a comprehensive overview of protocols for establishing and maintaining zebrafish embryonic cell lines, emphasizing culture conditions, pluripotency features, transfection strategies, and recent innovations such as genotype-defined mutant lines generated via CRISPR/Cas9 and feeder-free systems. We also highlight emerging applications in oncology, regenerative medicine, and functional genomics, positioning zebrafish cell lines as versatile platforms bridging animal models and next-generation in vitro systems. Its continued optimization holds promise for improved reproducibility, reduced animal use, and expanded translational impact in biomedical research. Full article