Copper chaperone antioxidant 1: multiple roles and a potential therapeutic target.

Copper (Cu) was recently demonstrated to play a critical role in cellular physiological and biochemical processes, including energy production and maintenance, antioxidation and enzymatic activity, and signal transduction. Antioxidant 1 (ATOX1), a chaperone of Cu previously named human ATX1 homologue (HAH1), has been found to play an indispensable role in maintaining cellular Cu homeostasis, antioxidative stress, and transcriptional regulation. In the past decade, it has also been found to be involved in a variety of diseases, including numerous neurodegenerative diseases, cancers, and metabolic diseases. Recently, increasing evidence has revealed that ATOX1 is involved in the regulation of cell migration, proliferation, autophagy, DNA damage repair (DDR), and death, as well as in organism development and reproduction. This review summarizes recent advances in the research on the diverse physiological and cytological functions of ATOX1 and the underlying mechanisms of its action in human health and diseases. The potential of ATOX1 as a therapeutic target is also discussed. This review aims to pose unanswered questions related to ATOX1 biology and explore the potential use of ATOX1 as a therapeutic target.

MiR-18 inhibitor promotes the differentiation of bovine skeletal muscle-derived satellite cells by increasing MEF2D expression.

Skeletal muscle is composed of muscle fibers formed from myoblast differentiation. Recently, numerous researchers have demonstrated that microRNAs (miRNAs) play an essential role in modulating the proliferation and differentiation of myoblasts. Our previous study has shown that among the miR-17-92 cluster members, miR-17 and miR-20a together with miR-19b can efficiently promote the differentiation of murine C2C12 and bovine primary myoblasts. However, the role of miR-18 in this process remains elusive. In this study, we revealed that miR-18 inhibited the differentiation of bovine skeletal muscle-derived satellite cells (bMDSCs), whereas an miR-18 inhibitor significantly promoted cell differentiation (p < 0.001). Then, a target gene of miR-18 was found to be myocyte enhancer factor 2D (MEF2D), which is critical for myoblast differentiation. Furthermore, we found that the combination of the miR-18 inhibitor and miR-19 significantly improved the formation of bMDSCs-derived muscle fibers (p < 0.001). This study revealed the role of miR-18 in bovine skeletal muscle differentiation and contributed to the understanding of the regulatory mechanism of mammalian myogenic differentiation.

Beef is a beneficial food source, and improving muscle yield and quality has become a hot topic in the beef industry. Therefore, our study aimed to explore effective methods to improve bovine muscle cell differentiation to increase beef production. The study revealed that microRNA-18 (miR-18) inhibitor could promote the differentiation of bovine skeletal muscle-derived satellite cells (bMDSCs) by increasing the expression of myocyte enhancer factor 2D (MEF2D), a critical gene for myoblast differentiation. Furthermore, we found that combined inhibitors of miR-18 and miR-19 could significantly improve bMDSCs differentiation. Our study demonstrated the role of a new regulatory factor that may enhance beef production level and contributed to elucidating the mechanism of muscle differentiation.

Study on Lgr5 expression in pancreas tissues and organoids by lineage tracing.

Leucine rich repeat containing G protein-coupled receptor 5(Lgr5) is widely expressed in multiple tissues and can be used as a stem cell marker in a variety of epithelial organs (including the small intestine, colon, stomach and hair follicles). In this study, we used Lgr5-CreERT2+/- and Rosa26-mTmG hybridized transgenic mice to investigate the expression of Lgr5 in both ductal epithelial cells during pancreas development and in vitro cultured pancreatic duct organoids. After induction with Tamoxifen, the Lgr5 expression was analyzed by detecting the enhanced green fluorescence protein in the pancreatic tissue sections in adult animals and embryos at different developmental stages. The results showed that Lgr5 expression was detected neither in adult pancreatic duct epithelia nor in the embryonic pancreatic tissues at day 15.5 or in newborn mice. However, when 4-hydroxy-Tamoxifen was supplemented to the culture medium, EGFP could be detected in the primary pancreatic duct organoids from Lgr5-Cre ERT2+/-; Rosa26-mTmG mice. These results suggested that Lgr5 was not expressed in adult and embryonic pancreatic tissues; but could be expressed in the cultured pancreas ductal organoids. The research lays the foundation for exploring specific gene expression patterns in stem/progenitor cells during pancreatic development.

A saponin from astragalus promotes pancreatic ductal organoids differentiation into insulin-producing cells.

BACKGROUND: Islet transplantation is an effective treatment for the type 1 and severe type 2 diabetes, but it is restricted by the severe lack of pancreas donors. In vitro differentiation of pancreatic progenitors into insulin-secreting cells is one of the hopeful strategies in the cell transplantation therapy of diabetes. Isoastragaloside I is one of the saponin molecules found in Astragalus membranaceus, which has been demonstrated to alleviate insulin resistance and glucose intolerance in obese mice. STUDY DESIGN: We established mouse pancreatic ductal organoids (mPDOs) with progenitor characteristics and an insulin promoter-driven EGFP reporter system to screen astragalus saponin components for monomers that can promote insulin-producing cell differentiation. METHODS: mPDOs treated with or without astragalus saponin monomers were investigated by the insulin promoter-driven EGFP reporter, quantitative PCR, immunofluorescence and flow cytometry to evaluate the expression of endocrine progenitor and ß-cell markers. RESULTS: Isoastragaloside I significantly promoted the expression of ß-cell differentiation genes, which was demonstrated by the activation of the insulin promoter-driven EGFP reporter, as well as the significant increase of mRNA levels of the endocrine progenitor marker Ngn3 and the ß-cell markers insulin1 and insulin2. Immunostaining studies indicated that the ß-cell-specific C-peptide was upregulated in isoastragaloside I-treated mPDOs. FACS analysis revealed that the ratio of C-peptide-secreting cells in isoastragaloside I-treated mPDOs was over 40%. Glucose tolerance tests demonstrated that the differentiated mPDOs could secrete C-peptide in response to glucose stimulation. CONCLUSIONS: We discover a novel strategy of inducing pancreatic ductal progenitors to differentiate into insulin-producing cells using isoastragaloside I. This approach can be potentially applied to ß-cell transplantation in diabetes therapies.

Copper enhances genotoxic drug resistance via ATOX1 activated DNA damage repair.

Copper is involved in various biochemical and physiological processes. The absorbed copper ions are transported to the intracellular destination via copper chaperones, such as ATOX1. Previous studies have demonstrated that neoplastic cells have a high demand for copper; however, its role in cancer cells has not been fully elucidated. Here, we reveal that the high level of copper contributes to drug resistance and repair of damaged DNA in cancer cells at least partially via ATOX1-induced expression of MDC1, a crucial protein involved in double-strand DNA damage repair. Specifically, ATOX1 enters into nuclear to target MDC1 promoter after treatments of various genotoxic agents, thus promoting the transcription of MDC1 in a copper-dependent manner. Therefore, knockout or blockage of ATOX1 conferred sensitivity to Gemcitabine in transplanted tumor mouse models. Together, our findings gain new insight into the role of copper in DNA damage repair and provide a novel strategy for clinical cancer therapy of drug-resistance cancers.

Comparative transcriptome analysis of rainbow trout gonadal cells (RTG-2) infected with U and J genogroup infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is the causative pathogen of infectious hematopoietic necrosis, outbreaks of which are responsible for significant losses in rainbow trout aquaculture. Strains of IHNV isolated worldwide have been classified into five major genogroups, J, E, L, M, and U. To date, comparative transcriptomic analysis has only been conducted individually for the J and M genogroups. In this study, we compared the transcriptome profiles in U genogroup and J genogroup IHNV-infected RTG-2 cells with mock-infected RTG-2 cells. The RNA-seq results revealed 17,064 new genes, of which 7,390 genes were functionally annotated. Differentially expressed gene (DEG) analysis between U and J IHNV-infected cells revealed 2,238 DEGs, including 1,011 downregulated genes and 1,227 upregulated genes. Among the 2,238 DEGs, 345 new genes were discovered. The DEGs related to immune responses, cellular signal transduction, and viral diseases were further analyzed. RT-qPCR validation confirmed that the changes in expression of the immune response-related genes trpm2, sting, itgb7, ripk2, and irf1, cellular signal transduction-related genes irl, cacnb2, bmp2l, gadd45α, and plk2, and viral disease-related genes mlf1, mtor, armc5, pik3r1, and c-myc were consistent with the results of transcriptome analysis. Taken together, our findings provide a comprehensive transcriptional analysis of the differential virulence of the U and J genogroups of IHNV, and shed new light on the pathogenic mechanisms of IHNV strains.

Establishment of a specific in vivo Cu(â ) reporting system based on metallothionein screening.

Copper is one of the indispensable trace metal elements in organisms, but excess copper means cytotoxicity. Cells protect themselves by storing excess copper in copper-binding proteins. Metallothioneins (MTs) are a group of low-molecular-weight, cysteine-rich proteins, which are well known for sensing and binding the overcharged Zn(Ⅱ), Cd(Ⅱ), and Cu(Ⅰ) in cells. However, there are only few reports on MTs that can specifically respond to intracellular copper ions in mammals in real-time. Here, we screened copper-response MTs in pancreatic cancer cells through data-mining, RNA-seq, and qPCR analysis. We found that MT1E, MT1F, and MT1X mRNA were significantly upregulated after exogenous copper ion induction. By constructing the stable cell lines with MT1E, MT1F, or MT1X promoter-driven EGFP as reporters, we found that only PMT1F-EGFP could specifically and stably report the intracellular Cu(Ⅰ) changes in multiple cell lines including Panc-1, 8988T, 293T, HepG2, and normal hepatic cells, indicating that PMT1F-EGFP is an ideal in vivo Cu(Ⅰ) reporter. Using the PMT1F-EGFP reporter, we found that MEK inhibitors (U0126) and Astragaloside IV could significantly increase intracellular copper ions. According to these results, PMT1F-EGFP reporter can sense intracellular copper change and can be used to screen copper-target drugs and study copper-related cellular physiology and pathology.

MiR-7 mediates mitochondrial impairment to trigger apoptosis and necroptosis in Rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS) is a pediatric cancer with rhabdomyoblastic phenotype and mitochondria act as pivotal regulators of its growth and progression. While miR-7-5p (miR-7) is reported to have a tumor-suppressive role, little is yet known about its antitumor activity in RMS. METHODS: The effects of miR-7 on RMS were analyzed both in vitro and in vivo. Cell death modalities induced by miR-7 were identified. Influence on mitochondria was evaluated through RNA sequencing data, morphological observation and mitochondrial functional assays, including outer membrane permeability, bioenergetics and redox balance. Dual-luciferase assay and phenotype validation after transient gene silencing were performed to identify miR-7 targets in RMS. RESULTS: MiR-7 executed anti-tumor effect in RMS beyond proliferation inhibition. Morphologic features and molecular characteristics with apoptosis and necroptosis were found in miR-7-transfected RMS cells. Chemical inhibitors of apoptosis and necroptosis were able to prevent miR-7-induced cell death. Further, we identified that mitochondrial impairment mainly contributed to these phenomena and mitochondrial proteins SLC25A37 and TIMM50 were crucial targets for miR-7 to induce cell death in RMS. CONCLUSION: Our results extended the mechanism of miR-7 antitumor role in rhabdomyosarcoma cancer, and provided potential implications for its therapy.

[Toxicity reduction of human islet amyloid polypeptide by Trim-Away technique in insulinoma cells].

Human islet amyloid polypeptide (hIAPP, also known as amylin) is a co-secreting protein of insulin in human pancreatic ß-cells. It is encapsulated in vesicles and secreted out of the cells with insulin. hIAPP can promote insulin secretion and regulate blood glucose homeostasis in the body under the normal physiological conditions. However, hIAPP misfolding or excessive accumulation can cause toxic effects on the ß cells, which in turn affect cell function, resulting in type 2 diabetes mellitus (T2DM) for the affected individuals. In order to eliminate the excessive accumulation of hIAPP in the cell and to maintain its normal synthetic function, we have adopted a new protein degradation technology called Trim-Away, which can degrade the target protein in a short time without affecting the mRNA transcription and translation synthesis function of the target protein. First, we overexpressed hIAPP in the rat insulinoma cells (INS1) to simulate its excessive accumulation and analyzed its effect in INS1 cells by measuring the release of LDH (lactate dehydrogenase), CCK8 activity and PI-Annexin V positive ratio. Results showed that excessive accumulation of hIAPP caused ß cell apoptosis. Second, real-time quantitative PCR analysis and ELISA detection showed that the synthesis and secretion of insulin were hindered. We used Trim-Way technology to specifically eliminate the excessive accumulation of hIAPP protein in hIAPP overexpressing INS1 cells. Cell activity experiments confirmed that clearance of hIAPP reduced the cell death phenotype. Further ELISA experiments confirmed that INS1 cells restored insulin secretion ability. This study examined the toxic effect of hIAPP excessive accumulation in INS1 cells and demonstrated the cytotoxicity clearance effect of Trim-Way technology in pancreatic ß-cells. Our research has provided a new strategy for using Trim-Away technology for treatment of diabetes.

Copper promotes sheep pancreatic duct organoid growth by activation of an antioxidant protein 1-dependent MEK-ERK pathway.

Proper amounts of copper supplemented in livestock feed improve the physical growth and traits of farm animals. The pancreas is an important organ with both exocrine and endocrine portions. To investigate the role and mechanism of copper in the sheep pancreas, we first established sheep pancreatic duct organoids (sPDOs). We found that an appropriate amount of copper benefited the formation and growth of sPDOs, whereas excess or deficient copper damaged sPDOs. We found that the proliferation-stimulating effect of copper was related to the copper chaperone antioxidant protein 1 (ATOX1)-dependent activation of MEK-ERK1/2 signaling. Atox1 knockdown suppressed the cell proliferation of sPDOs, even in the presence of the MEK activator. These results indicate that moderate concentrations of copper promote sPDO growth through ATOX1-regulated cell proliferation by activation of MEK-ERK. Moreover, our study indicates that organoids may be a useful model to study organ growth mechanisms in livestock.

EGF suppresses the expression of miR-124a in pancreatic ß cell lines via ETS2 activation through the MEK and PI3K signaling pathways.

Diabetes mellitus is characterized by pancreatic ß cell dysfunction. Previous studies have indicated that epidermal growth factor (EGF) and microRNA-124a (miR-124a) play opposite roles in insulin biosynthesis and secretion by beta cells. However, the underlying mechanisms remain poorly understood. In the present study, we demonstrated that EGF could inhibit miR-124a expression in beta cell lines through downstream signaling pathways, including mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) cascades. Further, the transcription factor ETS2, a member of the ETS (E26 transformation-specific) family, was identified to be responsible for the EGF-mediated suppression of miR-124a expression, which was dependent on ETS2 phosphorylation at threonine 72. Activation of ETS2 decreased miR-124a promoter transcriptional activity through the putative conserved binding sites AGGAANA/TN in three miR-124a promoters located in different chromosomes. Of note, ETS2 played a positive role in regulating beta cell function-related genes, including miR-124a targets, Forkhead box a2 (FOXA2) and Neurogenic differentiation 1 (NEUROD1), which may have partly been through the inhibition of miR-124 expression. Knockdown and overexpression of ETS2 led to the prevention and promotion of insulin biosynthesis respectively, while barely affecting the secretion ability. These results suggest that EGF may induce the activation of ETS2 to inhibit miR-124a expression to maintain proper beta cell functions and that ETS2, as a novel regulator of insulin production, is a potential therapeutic target for diabetes mellitus treatment.

Common Carp mef2 Genes: Evolution and Expression.

The MEF2 (myocyte enhancer factor 2) family belongs to the MADS-box superfamily of eukaryotic transcription factors. The vertebrate genes compose four distinct subfamilies designated MEF2A, -B, -C, and -D. There are multiple mef2 genes in the common carp (Cyprinus carpio). So far, the embryonic expression patterns of these genes and the evolution of fish mef2 genes have been barely investigated. In this study, we completed the coding information of C. carpio mef2ca2 and mef2d1 genes via gene cloning and presented two mosaic mef2 sequences as evidence for recombination. We also analyzed the phylogenetic relationship and conserved synteny of mef2 genes and proposed a new evolutionary scenario. In our version, MEF2B and the other three vertebrate subfamilies were generated in parallel from the single last ancestor via two rounds of whole genome duplication events that occurred at the dawn of vertebrates. Moreover, we examined the expression patterns of C. carpio mef2 genes during embryogenesis, by using whole-mount in situ hybridization, and found the notochord to be a new expression site for these genes except for mef2ca1&2. Our results thus provide new insights into the evolution and expression of mef2 genes.

MiR-17 and miR-19 cooperatively promote skeletal muscle cell differentiation.

Skeletal myogenesis is a highly coordinated process that involves cell proliferation, differentiation and fusion controlled by a complex gene regulatory network. The microRNA gene cluster miR-17-92 has been shown to be related to this process; however, the exact role of each cluster member remains unclear. Here, we show that miR-17 and miR-20a could effectively promote the differentiation of both C2C12 myoblasts and primary bovine satellite cells. In contrast, miR-18a might play a negative role in C2C12 cell differentiation, while miR-19 and miR-92a had little influence. Transcriptome and target analyses revealed that miR-17 could act on Ccnd2, Jak1 and Rhoc genes that are critical for cell proliferation and/or fusion. Notably, the addition of miR-19 could reverse the lethal effect of miR-17 and could thus facilitate the maturation of myotubes. Furthermore, by co-injecting the lentiviral shRNAs of miR-17 and miR-19 into mouse tibialis anterior muscles, we demonstrated the wound healing abilities of the two miRNAs. Our findings indicate that in combination with miR-19, miR-17 is a potent inducer of skeletal muscle differentiation.

Guanine-rich RNA binding protein GRSF1 inhibits myoblast differentiation through repressing mitochondrial ROS production.

Guanine-rich RNA sequence binding factor 1 (GRSF1) is a member of the RNA-binding protein (RBP) family. GRSF1 regulates RNA metabolism through RNA processing, transport and translation in the cytoplasm and mitochondria. However, its role in myogenesis has not been investigated. Here, we demonstrated that the expression of mitochondrial GRSF1 was negatively related to the differentiation of mouse skeletal myoblasts. Interference with GRSF1 promotes myogenesis both in vitro and in vivo without affecting MyoD expression or cell proliferation. Further studies illustrated that GRSF1 regulated myogenic differentiation through direct targeting of mitochondrial GPX4, a key regulator of the cellular redox status, leading to the modulation of ROS levels, which is important for myogenesis. Our findings underscore a critical function for GRSF1 during skeletal myogenesis linked to its regulation of muscle redox homeostasis.

Blockage of SLC31A1-dependent copper absorption increases pancreatic cancer cell autophagy to resist cell death.

OBJECTIVES: Clinical observations have demonstrated that copper levels elevate in several cancer types, and copper deprivation is shown to inhibit tumour angiogenesis and growth in both animal models and preclinical trials. However, the content of copper in pancreatic duct adenocarcinoma (PDAC) and whether it is a potential therapy target is still unknown. MATERIALS AND METHODS: The levels of copper in PDAC specimens were detected by ICP-MS assays. Copper depletion in Panc-1 or MiaPaCa-2 cells was conducted via copper transporter 1 (SLC31A1) interference and copper chelator tetrathiomolybdate (TM) treatment. The effects of copper deprivation on cancer cells were evaluated by cell proliferation, migration, invasion, colony formation and cell apoptosis. The mechanism of copper deprivation-caused cancer cell quiescence was resolved through mitochondrial dysfunction tests and autophagy studies. The tumour-suppression experiments under the condition of copper block and/or autophagy inhibition were performed both in vitro and in xenografted mice. RESULTS: SLC31A1-dependent copper levels are correlated with the malignant degree of pancreatic cancer. Blocking copper absorption could inhibit pancreatic cancer progression but did not increase cell death. We found that copper deprivation increased mitochondrial ROS level and decreased ATP level, which rendered cancer cells in a dormant state. Strikingly, copper deprivation caused an increase in autophagy to resist death of pancreatic cancer cells. Simultaneous treatment with TM and autophagy inhibitor CQ increased cell death of cancer cells in vitro and retarded cancer growth in vivo. CONCLUSIONS: These findings reveal that copper deprivation-caused cell dormancy and the increase in autophagy is a reason for the poor clinical outcome obtained from copper depletion therapies for cancers. Therefore, the combination of autophagy inhibition and copper depletion is potentially a novel strategy for the treatment of pancreatic cancer and other copper-dependent malignant tumours.

Mutual inhibitions between epidermal growth factor receptor signaling and miR-124a control pancreatic progenitor proliferation.

Pancreatic stem/progenitor cells convert from a proliferative to a differentiated fate passing through proliferation cease to a resting state. However, the molecular mechanisms of cell cycle arrest are poorly understood. In this study, we demonstrated that the microRNA-124a (miR-124a) inhibited the proliferation of pancreatic progenitor cells both in vitro and ex vivo and promoted a quiescent state. The miR-124a directly targeted SOS Ras/Rac guanine nucleotide exchange factor 1 (SOS1), IQ motif-containing GTPase-activating protein 1 (IQGAP1), signal transducer and activator of transcription 3 (STAT3), and cyclin D2 (CCND2), thereby inactivating epidermal growth factor receptor (EGFR) downstream signaling pathways including mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK), phosphatidylinositol 3-kinase-protein kinase B (PI3K/AKT) and Janus kinase (JAK)/STAT3. miR-124a blocked cell proliferation mainly through targeting STAT3 to inhibit PI3K/AKT and JAK/STAT3 signaling. Moreover, miR-124a expression was negatively regulated by EGFR downstream PI3K/AKT signaling. These results indicated that miR-124a and EGFR signaling mutually interact to form a regulating circuit that determines the proliferation of pancreatic progenitor cells.

Baicalein inhibits pancreatic cancer cell proliferation and invasion via suppression of NEDD9 expression and its downstream Akt and ERK signaling pathways.

Baicalein, a flavone ingredient of Scutellaria baicalensis Georgi, is a promising anti-cancer agent. However, its potential anti-pancreatic cancer effects and the underlying mechanisms are still unclear. In this study, we showed that Baicalein not only induced apoptosis, but also suppressed proliferation, migration and invasion of two pancreatic cancer cell lines BxPC-3 and PANC-1 in a dose- and time-dependent manner. Notably, Baicalein exhibited low toxicity to normal human liver or kidney cells. We further discovered that Baicalein suppressed BxPC-3 and PANC-1 cell proliferation and invasion through targeting the expression of NEDD9, a Cas scaffolding protein, to decrease Akt and ERK activities. Especially, Baicalein decreased Akt phosphorylation at T-308 via lowering NEDD9-dependent PDK1 expression. Overexpression of NEDD9 effectively rescued proliferation and invasion of BxPC-3 and PANC-1 cells dampened by Baicalein. Taken together, our findings suggest that Baicalein is a potent remedy applied to pancreatic cancer treatment in the future.

miR-18a counteracts AKT and ERK activation to inhibit the proliferation of pancreatic progenitor cells.

Activation of endogenous stem/progenitor cells to repair injured tissues is an ideal option for disease treatment. However, adult pancreatic progenitor cells remain in a quiescent state in vivo. Thus, it is difficult to stimulate proliferation and differentiation in these progenitor cells, and the cause remains elusive. miR-17-92 cluster miRNAs are highly conserved in mammals and are expressed in multiple tissue stem/progenitor cells, but their role in pancreatic progenitor cells are less well known. In the present study, we demonstrate that miR-18a, but not the other members of the miR-17-92 gene cluster, inhibits the proliferation of pancreatic progenitor cells in vitro and ex vivo. miR-18a inhibits proliferation of adult pancreatic progenitor cells through arresting the cell cycle at G1 stage, indicating that miR-18a plays a role in keeping the adult pancreatic progenitor cells in quiescence. miR-18a inhibits pancreatic progenitor proliferation by targeting the gene expressions of connective tissue growth factor (CTGF), neural precursor cell expressed, developmentally down-regulated 9 (Nedd9), and cyclin dependent kinase 19 (CDK19), as well as by suppressing activation of the proliferation-related signaling pathways phosphatidylinositol 3-kinase-protein kinase B (PI3K/AKT) and extracellular signal-regulated kinase (ERK).

Inhibiting cyprinid herpesvirus-3 replication with CRISPR/Cas9.

OBJECTIVES: The potential of CRISPR/Cas9 gene editing to repress CyHV-3 was tested in vitro. RESULTS: By targeting two basic target genes necessary for the early transcription of CyHV-3, we show that virus transcription and particle release were significantly decreased by CRISPR/Cas9, as measured by quantitative real-time PCR and virus titration experiments, respectively. CONCLUSIONS: (A) The effectiveness is confirmed of the CRISPR/Cas9 system at repressing exogenous genes, including large viral genomic DNA, by introducing site-specific mutations in vitro. (B) The CyHV-3 virus replicates poorly in Cas9-positive cells. (C) The inhibition of thymidine kinase alone cannot block viral particle release.