Breaking the barrier: Epigenetic strategies to combat platinum resistance in colorectal cancer.

Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide. Platinum-based drugs, such as cisplatin and oxaliplatin, are frontline chemotherapy for CRC, effective in both monotherapy and combination regimens. However, the clinical efficacy of these treatments is often undermined by the development of drug resistance, a significant obstacle in cancer therapy. In recent years, epigenetic alterations have been recognized as key players in the acquisition of resistance to platinum drugs. Targeting these dysregulated epigenetic mechanisms with small molecules represents a promising therapeutic strategy. This review explores the complex relationship between epigenetic changes and platinum resistance in CRC, highlighting current epigenetic therapies and their effectiveness in countering resistance mechanisms. By elucidating the epigenetic underpinnings of platinum resistance, this review aims to contribute to ongoing efforts to improve treatment outcomes for CRC patients.

Targeting the Tumor Epigenetic Regulator SETDB1 for Tumor Therapy.

Epigenetic alterations are implicated in the early stages of tumorigenesis and are widely recognized as a ubiquitous phenomenon in cancer development. Aberrant epigenetic modifications can alter the expression of target genes, induce heterochromatin formation, and gradually drive normal cells towards immortalized tumor cells with significant consequences. SETDB1 (SET domain bifurcated histone lysine methyltransferase 1), a typical histone me-thyltransferase, promotes the formation of heterochromatin and inhibits the transcription of genes by modifying the methylation of lysine 9 of histone 3. SETDB1 is usually highly ex-pressed in tumors with high copy numbers, accompanied by poor prognosis and low patient survival rates, which is a typical case of abnormal epigenetic modification. We discuss the mechanism of SETDB1 in a variety of cancers and review the epigenetic inhibitors that have been reported in recent years, along with their anti-tumor effects. In addition, we summarize the role of SETDB1 in a variety of diseases and cell functions.

Unraveling the nexus of age, epilepsy, and mitochondria: exploring the dynamics of cellular energy and excitability.

Epilepsy, a complex neurological condition marked by recurring seizures, is increasingly recognized for its intricate relationship with mitochondria, the cellular powerhouses responsible for energy production and calcium regulation. This review offers an in-depth examination of the interplay between epilepsy, mitochondrial function, and aging. Many factors might account for the correlation between epilepsy and aging. Mitochondria, integral to cellular energy dynamics and neuronal excitability, perform a critical role in the pathophysiology of epilepsy. The mechanisms linking epilepsy and mitochondria are multifaceted, involving mitochondrial dysfunction, reactive oxygen species (ROS), and mitochondrial dynamics. Mitochondrial dysfunction can trigger seizures by compromising ATP production, increasing glutamate release, and altering ion channel function. ROS, natural byproducts of mitochondrial respiration, contribute to oxidative stress and neuroinflammation, critical factors in epileptogenesis. Mitochondrial dynamics govern fusion and fission processes, influence seizure threshold and calcium buffering, and impact seizure propagation. Energy demands during seizures highlight the critical role of mitochondrial ATP generation in maintaining neuronal membrane potential. Mitochondrial calcium handling dynamically modulates neuronal excitability, affecting synaptic transmission and action potential generation. Dysregulated mitochondrial calcium handling is a hallmark of epilepsy, contributing to excitotoxicity. Epigenetic modifications in epilepsy influence mitochondrial function through histone modifications, DNA methylation, and non-coding RNA expression. Potential therapeutic avenues targeting mitochondria in epilepsy include mitochondria-targeted antioxidants, ketogenic diets, and metabolic therapies. The review concludes by outlining future directions in epilepsy research, emphasizing integrative approaches, advancements in mitochondrial research, and ethical considerations. Mitochondria emerge as central players in the complex narrative of epilepsy, offering profound insights and therapeutic potential for this challenging neurological disorder.

Molecular mechanisms for DNA methylation defects induced by ICF syndrome-linked mutations in DNMT3B.

DNA methyltransferase 3B (DNMT3B) plays a crucial role in DNA methylation during mammalian development. Mutations in DNMT3B are associated with human genetic diseases, particularly immunodeficiency, centromere instability, facial anomalies (ICF) syndrome. Although ICF syndrome-related missense mutations in the DNMT3B have been identified, their precise impact on protein structure and function remains inadequately explored. Here, we delve into the impact of four ICF syndrome-linked mutations situated in the DNMT3B dimeric interface (H814R, D817G, V818M, and R823G), revealing that each of these mutations compromises DNA-binding and methyltransferase activities to varying degrees. We further show that H814R, D817G, and V818M mutations severely disrupt the proper assembly of DNMT3B homodimer, whereas R823G does not. We also determined the first crystal structure of the methyltransferase domain of DNMT3B-DNMT3L tetrameric complex hosting the R823G mutation showing that the R823G mutant displays diminished hydrogen bonding interactions around T775, K777, G823, and Q827 in the protein-DNA interface, resulting in reduced DNA-binding affinity and a shift in sequence preference of +1 to +3 flanking positions. Altogether, our study uncovers a wide array of fundamental defects triggered by DNMT3B mutations, including the disassembly of DNMT3B dimers, reduced DNA-binding capacity, and alterations in flanking sequence preferences, leading to aberrant DNA hypomethylation and ICF syndrome.

o8G-miR-6513-5p/BCL2L13 Axis Regulates Mitophagy during Oxidative Stress in the Human Saphenous Vein Endothelial Cells.

Venous graft decay (VGD) occurs in coronary artery bypass grafting (CABG), and ischemia-reperfusion oxidative stress injury during the operation is involved in VGD. To explore the cellular phenotypic changes during this process, a stable oxidative stress model of human saphenous vein endothelial cells (HSVECs) is constructed. Through proteomics and cell experiments, it is found that the expression of BCL2L13 is upregulated during oxidative stress of HSVECs, and BCL2L13 regulated mitophagy through receptor-mediated interaction with LC3 and plays a role in cell protection. During oxidative stress, intracellular o8G epigenetic modification occurs, and the o8G modification of miR-6513-5p causes this molecule to lose its targeted regulation of BCL2L13 and participates in the upregulation of BCL2L13. There is a regulatory pathway of o8G modification-BCL2L13-LC3-mitophagy when oxidative stress occurs in HSVECs.

PSF-lncRNA interaction as a target for novel targeted anticancer therapies.

The Polypyrimidine Tract-Binding Protein-Associated Splicing Factor (PSF), a component of the Drosophila Behavior/Human Splicing (DBHS) complex, plays a pivotal role in cancer pathogenesis. The epigenetic regulation mediated by PSF and long noncoding RNA (lncRNA), along with PSF's alternative splicing activity, has been implicated in promoting cancer cell proliferation, migration, invasion, metastasis, and drug resistance in various human cancers. Recent research highlights the therapeutic promise of targeting the PSF-lncRNA interaction to combat aggressive malignancies, making it a compelling target for cancer therapy. This review offers a detailed synthesis of the current understanding of PSF's role in oncogenic pathways and recent progress in identifying inhibitors of PSF-lncRNA interactions. Furthermore, it discusses the potential of using these inhibitors in cancer treatment strategies, especially as adjuncts to immune checkpoint blockade therapies to improve the efficacy of anti-PD-(L)1 treatments in Glioblastoma Multiforme (GBM). By outlining the interaction patterns of existing PSF-lncRNA inhibitors, this article aims to guide the development and refinement of future pharmacological interventions.

Abnormal Weakening of DNA Methylation around the SLC6A1 Gene Promoter in Temporal Lobe Epilepsy.

BACKGROUND: The solute carrier (SLC) superfamily, which transports solutes across biological membranes, includes four members (SLC2A1, SLC6A1, SLC9A64, and SLC35A2) that have been linked to epilepsy. This study sought to examine the DNA methylation patterns near the promoters of these genes in temporal lobe epilepsy (TLE), as DNA methylation is a crucial epigenetic modification that can impact gene expression. METHODS: The study comprised 38 individuals with TLE and 38 healthy controls. Methylation experiments were performed using peripheral blood, while demethylation experiments were carried out using SH-SY5Y cells with the DNA methylation inhibitor decitabine. RESULTS: A significant difference was observed in the DNA methylation rate of SLC6A1 between TLE patients and controls, with TLE patients showing a lower rate (4.81% vs. 5.77%, p = 0.0000), which remained significant even after Bonferroni correction (p = 0.0000). Based on the hypomethylated SLC6A1 in TLE, a predictive model was established that showed promise in distinguishing and calibrating TLE. In the TLE group, there were differences in DNA methylation rates of SLC6A1 between the young patients and the older controls (4.42% vs. 5.22%, p = 0.0004). A similar trend (p = 0.0436) was noted after adjusting for sex, age at onset, and drug response. In addition, the study found that DNA methylation had a silencing impact on the expression of the SLC6A1 gene in SH-SY5Y cells, which were treated with decitabine at a set dose gradient. CONCLUSIONS: The evidence suggests that lower methylation of SLC6A1 may stimulate transcription in TLE, however, further investigation is necessary to confirm the exact mechanism.

Genetics, epigenetics and autoimmunity constitute a Bermuda triangle for the pathogenesis of rheumatoid arthritis.

Rheumatoid arthritis (RA), a multigene disorder with a heritability rate of 60 %, is characterized by persistent pain, synovial hyperplasia, and cartilage and bone destruction, ultimately causing irreversible joint deformity. The etiology and pathogenesis of rheumatoid arthritis (RA) are primarily influenced by specific genetic variants, particularly HLA alleles such as HLA-DRB1*01 and DRB1*04. However, other HLA alleles such as HLA-DRB1*10 and DPB*1 have also been found to contribute to increased susceptibility to RA. However, non-HLA genes also confer a comparatively high risk of RA disease manifestation. The most relevant single nucleotide polymorphisms (SNPs) associated with non-HLA genes are PTPN22, TRAF1, CXCL-12, TBX-5, STAT4, FCGR, PADI4, and MTHFR. In conjunction with genetic susceptibility, epigenetic alterations orchestrate paramount involvement in regulating RA pathogenesis. Increasing evidence implicates DNA methylation and histone protein modifications, including acetylation and methylation, as the primary epigenetic mechanisms that drive the pathogenesis and clinical progression of the disease. In addition to genetic and epigenetic changes, autoimmune inflammation also determines the pathological progression of the synovial membrane in joints with RA. Glycosylation changes, such as sialylation and fucosylation, in immune cells have been shown to be relevant to disease progression. Genetic heterogeneity, epigenetic factors, and changes in glycosylation do not fully explain the features of RA. Therefore, investigating the interplay between genetics, epigenetics, and autoimmunity is crucial. This review highlights the significance and interaction of these elements in RA pathophysiology, suggesting their diagnostic potential and opening new avenues for novel therapeutic approaches.

Host Risk Factors for Tuberculosis.

Tuberculosis (TB) spreads through droplets that contain Mycobacterium tuberculosis (Mtb) and can infect susceptible people. Due to different risk factors, people have different susceptibility ranges towards TB. The risk factors are classified into three main groups, includ-ing bacterial, environmental, and host factors. Literature review reveals that the most important host risk factors are aging, male gender, genetics, epigenetics, having an impaired immune system, diabetes, malignancy, malnutrition, anemia, and pregnancy. The risk factors contribute to the increase in TB cases through inflammation, increased contact with TB patients, disrup-tion of immune genes, changes in gene expression, increased activity of Mtb, damage to cellu-lar immunity, reactivation of Latent TB Infection (LTBI), increased susceptibility to TB, com-promised immunity, and changes in the proportion of T cell subgroups, respectively. Therefore, identification of the infection source and high-risk people and timely treatment of the patients can reduce TB mortality and help control the disease.

Krüppel-like factors family in health and disease.

Krüppel-like factors (KLFs) are a family of basic transcription factors with three conserved Cys2/His2 zinc finger domains located in their C-terminal regions. It is acknowledged that KLFs exert complicated effects on cell proliferation, differentiation, survival, and responses to stimuli. Dysregulation of KLFs is associated with a range of diseases including cardiovascular disorders, metabolic diseases, autoimmune conditions, cancer, and neurodegenerative diseases. Their multidimensional roles in modulating critical pathways underscore the significance in both physiological and pathological contexts. Recent research also emphasizes their crucial involvement and complex interplay in the skeletal system. Despite the substantial progress in understanding KLFs and their roles in various cellular processes, several research gaps remain. Here, we elucidated the multifaceted capabilities of KLFs on body health and diseases via various compliable signaling pathways. The associations between KLFs and cellular energy metabolism and epigenetic modification during bone reconstruction have also been summarized. This review helps us better understand the coupling effects and their pivotal functions in multiple systems and detailed mechanisms of bone remodeling and develop potential therapeutic strategies for the clinical treatment of pathological diseases by targeting the KLF family.

Downregulation of IL-8 and IL-10 by LRRC8A Inhibition through the NOX2-Nrf2-CEBPB Transcriptional Axis in THP-1-Derived M2 Macrophages.

M2-polarized, tumor-associated macrophages (TAMs) produce pro-tumorigenic and angiogenic mediators, such as interleukin-8 (IL-8) and IL-10. Leucine-rich repeat-containing protein 8 members (LRRC8s) form volume-regulated anion channels and play an important role in macrophage functions by regulating cytokine and chemokine production. We herein examined the role of LRRC8A in IL-8 and IL-10 expression in THP-1-differentiated M2-like macrophages (M2-MACs), which are a useful tool for investigating TAMs. In M2-MACs, the pharmacological inhibition of LRRC8A led to hyperpolarizing responses after a transient depolarization phase, followed by a slight elevation in the intracellular concentration of Ca2+. Both the small interfering RNA-mediated and pharmacological inhibition of LRRC8A repressed the transcriptional expression of IL-8 and IL-10, resulting in a significant reduction in their secretion. The inhibition of LRRC8A decreased the nuclear translocation of phosphorylated nuclear factor-erythroid 2-related factor 2 (Nrf2), while the activation of Nrf2 reversed the LRRC8A inhibition-induced transcriptional repression of IL-8 and IL-10 in M2-MACs. We identified the CCAAT/enhancer-binding protein isoform B, CEBPB, as a downstream target of Nrf2 signaling in M2-MACs. Moreover, among several upstream candidates, the inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) suppressed the Nrf2-CEBPB transcriptional axis in M2-MACs. Collectively, the present results indicate that the inhibition of LRRC8A repressed IL-8 and IL-10 transcription in M2-MACs through the NOX2-Nrf2-CEBPB axis and suggest that LRRC8A inhibitors suppress the IL-10-mediated evasion of tumor immune surveillance and IL-8-mediated metastasis and neovascularization in TAMs.

Bcl-xL is translocated to the nucleus via CtBP2 to epigenetically promote metastasis.

Nuclear Bcl-xL is found to promote cancer metastasis independently of its mitochondria-based anti-apoptotic activity. How Bcl-xL is translocated into the nucleus and how nuclear Bcl-xL regulates histone H3 trimethyl Lys4 (H3K4me3) modification have yet to be understood. Here, we report that C-terminal Binding Protein 2 (CtBP2) binds to Bcl-xL via its N-terminus and translocates Bcl-xL into the nucleus. Knockdown of CtBP2 by shRNA decreases the nuclear portion of Bcl-xL and reverses Bcl-xL-induced invasion and metastasis in mouse models. Furthermore, knockout of CtBP2 not only reduces the nuclear portion of Bcl-xL but also suppresses Bcl-xL transcription. The binding between Bcl-xL and CtBP2 is required for their interaction with MLL1, a histone H3K4 methyltransferase. Pharmacologic inhibition of the MLL1 enzymatic activity reverses Bcl-xL-induced H3K4me3 and TGFß mRNA upregulation, as well as invasion. Moreover, the cleavage under targets and release using nuclease (CUT&RUN) assay coupled with next-generation sequencing reveals that H3K4me3 modifications are particularly enriched in the promotor regions of genes encoding TGFß and its signaling pathway members in cancer cells overexpressing Bcl-xL. Altogether, the metastatic function of Bcl-xL is mediated by its interaction with CtBP2 and MLL1 and this study offers new therapeutic strategies to treat Bcl-xL-overexpressing cancer.

Methylation modification of non-histone proteins in breast cancer: An emerging targeted therapeutic strategy.

Breast cancer is a major public health concern worldwide, being the most commonly diagnosed cancer among women and a leading cause of cancer-related deaths. Recent studies have highlighted the significance of non-histone methylation in breast cancer, which modulates the activity, interaction, localization, and stability of target proteins. This regulation affects critical processes such as oncogenesis, tumor growth, proliferation, invasion, migration, and immune responses. This review delves into the enzymes responsible for non-histone methylation, such as protein arginine methyltransferases (PRMTs), lysine methyltransferases (KMTs), and demethylases, and explores their roles in breast cancer. By elucidating the molecular mechanisms and functional consequences of non-histone methylation, this review aims to provide insights into novel therapeutic strategies targeting these pathways. The therapeutic potential of targeting non-histone methylation to overcome drug resistance and enhance treatment efficacy in breast cancer is also discussed, highlighting promising avenues for future research and clinical applications.

Insights into a functional synthetic plant genome.

Synthetic genomics involves the design, assembly, and transfer of artificially synthesized DNA fragments into target hosts to replace the native genome and construct viable forms of life. With advances in DNA synthesis and assembly techniques, the application of synthetic genomics in viruses, bacteria, and yeast has improved our knowledge of genome organization and function. Multicellular eukaryotic organisms are characterized by larger genomes, more complex epigenetic regulation, and widespread transposable elements, making genome synthesis challenging. Recently, the first synthetic multicellular eukaryotic organism was generated in the model plant Physcomitrium patens with a partially synthetic chromosome arm. Here, we introduce the design and assembly principles of moss genome synthesis. We also discuss the remaining technical barriers in the application of synthetic genomics in seed plants.

Epigenetic regulation of diverse regulated cell death modalities in cardiovascular disease: Insights into necroptosis, pyroptosis, ferroptosis, and cuproptosis.

Cell death constitutes a critical component of the pathophysiology of cardiovascular diseases. A growing array of non-apoptotic forms of regulated cell death (RCD)-such as necroptosis, ferroptosis, pyroptosis, and cuproptosis-has been identified and is intimately linked to various cardiovascular conditions. These forms of RCD are governed by genetically programmed mechanisms within the cell, with epigenetic modifications being a common and crucial regulatory method. Such modifications include DNA methylation, RNA methylation, histone methylation, histone acetylation, and non-coding RNAs. This review recaps the roles of DNA methylation, RNA methylation, histone modifications, and non-coding RNAs in cardiovascular diseases, as well as the mechanisms by which epigenetic modifications regulate key proteins involved in cell death. Furthermore, we systematically catalog the existing epigenetic pharmacological agents targeting novel forms of RCD and their mechanisms of action in cardiovascular diseases. This article aims to underscore the pivotal role of epigenetic modifications in precisely regulating specific pathways of novel RCD in cardiovascular diseases, thus offering potential new therapeutic avenues that may prove more effective and safer than traditional treatments.

Effects of Glycine on epigenetic modification and early embryonic development in porcine oocytes exposed to monobutyl phthalate.

Monobutyl phthalate (MBP) is the primary active metabolite of dibutyl phthalate (DBP), the key plasticizer component. A substantial body of evidence from studies conducted on both animals and humans indicates that MBP exposure could result in harmful impacts on toxicity pathways. In addition, it can seriously affect human and animal reproductive health. In our present study, we showed that exposure to MBP causes abnormal epigenetic modifications in porcine oocytes and failure of early embryonic development. However, glycine (Gly) can protect oocytes and early embryos from damage caused by MBP. Our study indicated a significant decrease in the percentage of porcine oocytes that reached the metaphase II (MII) phase when exposed to MBP. SET-domain-containing 2(SETD2)-mediated H3K36me3 histone methylation was detected, and the results showed that MBP significantly decreased the protein expression of H3K36me3 and SETD2. Moreover, the expression of the DNA break markers γH2AX and the mRNA expression of Asf1a, and Asf1b increased in the MBP group. The detection of DNA methylation marker proteins showed that MBP significantly increased the fluorescence intensity of 5-methylcytosine (5mC). The results from our RT-qPCR analysis demonstrated a significant decrease in the mRNA expression of the DNA methylation-related genes Dnmt1 and Dnmt3a, as well as the embryonic developmental potential-related genes Oct4 and Nanog, in porcine oocytes following exposure to MBP. Additionally, the mRNA expression of p53 significantly increased. Subsequently, the effects of MBP on early embryonic development were examined via parthenogenesis activation (PA) and in vitro fertilization (IVF). Exposure to MBP significantly impacted the development of embryos in both PA and IVF processes. The TUNEL staining data showed that MBP significantly increased embryonic apoptosis. However, Gly can ameliorate MBP-induced defects in oocyte epigenetic modifications and early embryonic development.

AQP1 differentially orchestrates endothelial cell senescence.

Accumulation of senescent endothelial cells (ECs) with age is a pivotal driver of cardiovascular diseases in aging. However, little is known about the mechanisms and signaling pathways that regulate EC senescence. In this report, we delineate a previously unrecognized role of aquaporin 1 (AQP1) in orchestrating extracellular hydrogen peroxide (H2O2)-induced cellular senescence in aortic ECs. Our findings underscore AQP1's differential impact on senescence hallmarks, including cell-cycle arrest, senescence-associated secretory phenotype (SASP), and DNA damage responses, intricately regulating angiogenesis. In proliferating ECs, AQP1 is crucial for maintaining angiogenic capacity, whereas disruption of AQP1 induces morphological and mitochondrial alterations, culminating in senescence and impaired angiogenesis. Conversely, Aqp1 knockdown or selective blockade of AQP1 in senescent ECs rescues the excess H2O2-induced cellular senescence phenotype and metabolic dysfunction, thereby ameliorating intrinsic angiogenic incompetence. Mechanistically, AQP1 facilitates H2O2 transmembrane transport, exacerbating oxidant-sensitive kinases CaMKII-AMPK. This process suppresses HDAC4 translocation, consequently de-repressing Mef2A-eNOS signaling in proliferating ECs. However, in senescent ECs, AQP1 overexpression is linked to preserved HDAC4-Mef2A complex and downregulation of eNOS signaling. Together, our studies identify AQP1 as a novel epigenetic regulator of HDAC4-Mef2A-dependent EC senescence and angiogenic potential, highlighting its potential as a therapeutic target for antagonizing age-related cardiovascular diseases.

Multi-faceted regulation of CREB family transcription factors.

cAMP response element-binding protein (CREB) is a ubiquitously expressed nuclear transcription factor, which can be constitutively activated regardless of external stimuli or be inducibly activated by external factors such as stressors, hormones, neurotransmitters, and growth factors. However, CREB controls diverse biological processes including cell growth, differentiation, proliferation, survival, apoptosis in a cell-type-specific manner. The diverse functions of CREB appear to be due to CREB-mediated differential gene expression that depends on cAMP response elements and multi-faceted regulation of CREB activity. Indeed, the transcriptional activity of CREB is controlled at several levels including alternative splicing, post-translational modification, dimerization, specific transcriptional co-activators, non-coding small RNAs, and epigenetic regulation. In this review, we present versatile regulatory modes of CREB family transcription factors and discuss their functional consequences.

Expression patterns and clinical value of key m6A RNA modification regulators in smoking patients with coronary artery disease.

Even though N6-methyladenosine (m6A) RNA modifications are increasingly being implicated in human disease, their mechanisms are not fully understood in smokers with coronary artery disease (CAD). Thirty m6A-related regulators' expression (MRRE) in CAD individuals (smokers and non-smokers) were analyzed from GEO. Support Vector Machine, random forest, and nomogram models were constructed to assess its clinical value. Consensus clustering, principal component analysis, and ssGSEA were used to construct a full picture of m6A-related regulators in smokers with CAD. Oxygen-glucose deprivation (OGD) and qRT-PCR were used to validate hypoxia's effect on MRRE. A comparison between smokers with CAD and controls revealed lower expression levels of RBM15B, YTHDC2, and ZC3H13. Based on three key MRREs, all models showed good clinical value, and smokers with CAD were divided into two distinct molecular subgroups. The correlations were found between key MRRE and the degree of immune infiltration. Three key MRREs in HUVECs and FMC84 mouse cardiomyocytes were reduced in the OGD group. Through hypoxia, smoking might reduce the expression levels of RBM15B, YTHDC2, and ZC3H13 in smokers with CAD. Our findings provide an important theoretical basis for the treatment of smokers with CAD.

Functional analysis of FlbA-regulated transcription factor genes in Aspergillus niger using a multiplexed CRISPRoff system.

FlbA of Aspergillus niger (indirectly) regulates 36 transcription factor (TF) genes. As a result, it promotes sporulation and represses vegetative growth, protein secretion and lysis. In this study, the functions of part of the FlbA-regulated TF genes were studied by using CRISPRoff. This system was recently introduced as an epigenetic tool for modulating gene expression in A. niger. A plasmid encompassing an optimized CRISPRoff system as well as a library of sgRNA genes that target the promoters of the 36 FlbA-regulated TF genes was introduced in A. niger. Out of 24 transformants that exhibited a sporulation phenotype, 12 and 18 strains also showed a biomass and secretion phenotype, respectively. The transforming sgRNAs, and thus the genes responsible for the phenotypes, were identified from five of the transformants. The results show that the genes dofA, dofB, dofC, dofD, and socA are involved in sporulation and extracellular enzyme activity, while dofA and socA also play roles in biomass formation. Overall, this study shows that the multiplexed CRISPRoff system can be effectively used for functional analysis of genes in a fungus.