ABSTRACT
Single-molecule techniques are ideally poised to characterize complex dynamics but are typically limited to investigating a small number of different samples. However, a large sequence or chemical space often needs to be explored to derive a comprehensive understanding of complex biological processes. Here we describe multiplexed single-molecule characterization at the library scale (MUSCLE), a method that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of complex dynamics. We comprehensively profiled the sequence dependence of DNA hairpin properties and Cas9-induced target DNA unwinding-rewinding dynamics. The ability to explore a large sequence space for Cas9 allowed us to identify a number of target sequences with unexpected behaviors. We envision that MUSCLE will enable the mechanistic exploration of many fundamental biological processes.
Subject(s)
DNA , High-Throughput Nucleotide Sequencing , Microscopy, Fluorescence , Single Molecule Imaging , High-Throughput Nucleotide Sequencing/methods , Single Molecule Imaging/methods , DNA/chemistry , DNA/genetics , Microscopy, Fluorescence/methods , CRISPR-Associated Protein 9 , Sequence Analysis, DNA/methods , Gene Library , CRISPR-Cas SystemsABSTRACT
The Ebola virus (EBOV) has emerged as a significant global health concern, notably during the 2013-2016 outbreak in West Africa. Despite the clinical approval of two EBOV antibody drugs, there is an urgent need for more diverse and effective antiviral drugs, along with comprehensive understanding of viral-host interactions. In this study, we harnessed a biologically contained EBOVΔVP30-EGFP cell culture model which could recapitulate the entire viral life cycle, to conduct a genome-wide CRISPR/Cas9 screen. Through this, we identified PIK3C3 (phosphatidylinositide 3-kinase) and SLC39A9 (zinc transporter) as crucial host factors for EBOV infection. Genetic depletion of SLC39A9 and PIK3C3 lead to reduction of EBOV entry, but not impact viral genome replication, suggesting that SLC39A9 and PIK3C3 act as entry factors, facilitating viral entry into host cells. Moreover, PIK3C3 kinase activity is indispensable for the internalization of EBOV virions, presumably through the regulation of endocytic and autophagic membrane traffic, which has been previously recognized as essential for EBOV internalization. Notably, our study demonstrated that PIK3C3 kinase inhibitor could effectively block EBOV infection, underscoring PIK3C3 as a promising drug target. Furthermore, biochemical analysis showed that recombinant SLC39A9 protein could directly bind viral GP protein, which further promotes the interaction of viral GP protein with cellular receptor NPC1. These findings suggests that SLC39A9 plays dual roles in EBOV entry. Initially, it serves as an attachment factor during the early entry phase by engaging with the viral GP protein. Subsequently, SLC39A9 functions an adaptor protein, facilitating the interaction between virions and the NPC1 receptor during the late entry phase, prior to cathepsin cleavage on the viral GP. In summary, this study offers novel insights into virus-host interactions, contributing valuable information for the development of new therapies against EBOV infection.
Subject(s)
CRISPR-Cas Systems , Ebolavirus , Hemorrhagic Fever, Ebola , Virus Internalization , Ebolavirus/genetics , Ebolavirus/physiology , Ebolavirus/metabolism , Humans , Hemorrhagic Fever, Ebola/virology , Hemorrhagic Fever, Ebola/metabolism , Hemorrhagic Fever, Ebola/genetics , Cation Transport Proteins/metabolism , Cation Transport Proteins/genetics , Class III Phosphatidylinositol 3-Kinases/metabolism , Class III Phosphatidylinositol 3-Kinases/genetics , Virus Replication , Animals , HEK293 CellsABSTRACT
Individuals with the Autism Susceptibility Candidate 2 (AUTS2) gene disruptions exhibit symptoms such as intellectual disability, microcephaly, growth retardation, and distinct skeletal and facial differences. The role of AUTS2 in neurodevelopment has been investigated using animal and embryonic stem cell models. However, the precise molecular mechanisms of how AUTS2 influences neurodevelopment, particularly in humans, are not thoroughly understood. Our study employed a 3D human cerebral organoid culture system, in combination with genetic, genomic, cellular, and molecular approaches, to investigate how AUTS2 impacts neurodevelopment through cellular signaling pathways. We used CRISPR/Cas9 technology to create AUTS2-deficient human embryonic stem cells and then generated cerebral organoids with these cells. Our transcriptomic analyses revealed that the absence of AUTS2 in cerebral organoids reduces the populations of cells committed to the neuronal lineage, resulting in an overabundance of cells with a transcription profile resembling that of choroid plexus (ChP) cells. Intriguingly, we found that AUTS2 negatively regulates the WNT/ß-catenin signaling pathway, evidenced by its overactivation in AUTS2-deficient cerebral organoids and in luciferase reporter cells lacking AUTS2. Importantly, treating the AUTS2-deficient cerebral organoids with a WNT inhibitor reversed the overexpression of ChP genes and increased the downregulated neuronal gene expression. This study offers new insights into the role of AUTS2 in neurodevelopment and suggests potential targeted therapies for neurodevelopmental disorders.
Subject(s)
Cell Differentiation , Cytoskeletal Proteins , Neurons , Organoids , Transcription Factors , Wnt Signaling Pathway , Humans , Organoids/metabolism , Neurons/metabolism , Transcription Factors/metabolism , Transcription Factors/genetics , Cytoskeletal Proteins/metabolism , Cytoskeletal Proteins/genetics , CRISPR-Cas Systems , beta Catenin/metabolism , beta Catenin/genetics , Human Embryonic Stem Cells/metabolism , Brain/metabolism , Brain/pathologyABSTRACT
Introduction: Mycoplasma pneumoniae (MP) is the major cause of respiratory infections that threaten the health of children and adolescents worldwide. Therefore, an early, simple, and accurate detection approach for MP is critical to prevent outbreaks of MP-induced community-acquired pneumonia. Methods: Here, we explored a simple and accurate method for MP identification that combines loop-mediated isothermal amplification (LAMP) with the CRISPR/Cas12b assay in a one-pot reaction. Results: In the current study, the whole reaction was completed within 1 h at a constant temperature of 57°C. The limit of detection of this assay was 33.7 copies per reaction. The specificity of the LAMP-CRISPR/Cas12b method was 100%, without any cross-reactivity with other pathogens. Overall, 272 clinical samples were used to evaluate the clinical performance of LAMP-CRISPR/Cas12b. Compared with the gold standard results from real-time PCR, the present method provided a sensitivity of 88.11% (126/143), specificity of 100% (129/129), and consistency of 93.75% (255/272). Discussion: Taken together, our preliminary results illustrate that the LAMP-CRISPR/Cas12b method is a simple and reliable tool for MP diagnosis that can be performed in resource-limited regions.
Subject(s)
CRISPR-Cas Systems , Molecular Diagnostic Techniques , Mycoplasma pneumoniae , Nucleic Acid Amplification Techniques , Pneumonia, Mycoplasma , Sensitivity and Specificity , Mycoplasma pneumoniae/genetics , Mycoplasma pneumoniae/isolation & purification , Nucleic Acid Amplification Techniques/methods , Humans , Pneumonia, Mycoplasma/diagnosis , Pneumonia, Mycoplasma/microbiology , Molecular Diagnostic Techniques/methods , Child , Limit of DetectionABSTRACT
The ZIKA virus (ZIKV) evades the host immune response by degrading STAT2 through its NS5 protein, thereby inhibiting type I interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism underlying this process has remained elusive. In this study, we performed a genome-wide CRISPR/Cas9 screen, revealing that ZSWIM8 as the substrate receptor of Cullin3-RING E3 ligase is required for NS5-mediated STAT2 degradation. Genetic depletion of ZSWIM8 and CUL3 substantially impeded NS5-mediated STAT2 degradation. Biochemical analysis illuminated that NS5 enhances the interaction between STAT2 and the ZSWIM8-CUL3 E3 ligase complex, thereby facilitating STAT2 ubiquitination. Moreover, ZSWIM8 knockout endowed A549 and Huh7 cells with partial resistance to ZIKV infection and protected cells from the cytopathic effects induced by ZIKV, which was attributed to the restoration of STAT2 levels and the activation of IFN signaling. Subsequent studies in a physiologically relevant model, utilizing human neural progenitor cells, demonstrated that ZSWIM8 depletion reduced ZIKV infection, resulting from enhanced IFN signaling attributed to the sustained levels of STAT2. Our findings shed light on the role of ZIKV NS5, serving as the scaffold protein, reprograms the ZSWIM8-CUL3 E3 ligase complex to orchestrate STAT2 proteasome-dependent degradation, thereby facilitating evasion of IFN antiviral signaling. Our study provides unique insights into ZIKV-host interactions and holds promise for the development of antivirals and prophylactic vaccines.
Subject(s)
Cullin Proteins , Interferon Type I , Proteolysis , STAT2 Transcription Factor , Signal Transduction , Ubiquitin-Protein Ligases , Ubiquitination , Viral Nonstructural Proteins , Zika Virus Infection , Zika Virus , Humans , STAT2 Transcription Factor/metabolism , Zika Virus/immunology , Zika Virus/physiology , Zika Virus/metabolism , Viral Nonstructural Proteins/metabolism , Viral Nonstructural Proteins/genetics , Interferon Type I/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Zika Virus Infection/metabolism , Zika Virus Infection/immunology , Zika Virus Infection/virology , Cullin Proteins/metabolism , A549 Cells , HEK293 Cells , CRISPR-Cas SystemsABSTRACT
The complex sample matrix poses significant challenges in accurately detecting heavy metals. In view of its superior performance for the biological adsorption of heavy metals, probiotic bacteria can be explored for functional unit to eliminate matrix interference. Herein, Lactobacillus rhamnosus (LGG) demonstrates a remarkable tolerance and can adsorb up to 300 µM of Hg2+, following the Freundlich isotherm model with the correlation coefficient (R2) value of 0.9881. Subsequently, by integrating the CRISPR/Cas12a system, a sensitive and specific fluorescent biosensor, "Cas12a-MB," has been developed for Hg2+ detection. Specifically, Hg2+ adsorbed onto LGG can specifically bind to the nucleic acid probe, thereby inhibiting the binding of the probe to LGG and the subsequent activation of the CRISPR/Cas12a system. Under optimal experimental conditions, with the detection time of 90 min and the detection limit of 0.44 nM, the "Cas12a-MB" biosensor offers a novel, eco-friendly approach for Hg2+ detection, showcasing the innovative application of probiotics in biosensor.
Subject(s)
Biosensing Techniques , CRISPR-Cas Systems , Lacticaseibacillus rhamnosus , Mercury , Probiotics , Mercury/analysis , Mercury/chemistry , Biosensing Techniques/methods , Probiotics/chemistry , Lacticaseibacillus rhamnosus/isolation & purification , Lacticaseibacillus rhamnosus/genetics , Adsorption , Limit of DetectionABSTRACT
With significant advancements in understanding gene functions and therapy, the potential misuse of gene technologies, particularly in the context of sports through gene doping (GD), has come to the forefront. This raises concerns regarding the need for point-of-care testing of various GD candidates to counter illicit practices in sports. However, current GD detection techniques, such as PCR, lack the portability required for on-site multiplexed detection. In this study, we introduce an integrated microfluidics-based chip for multiplexed gene doping detection, termed MGD-Chip. Through the strategic design of hydrophilic and hydrophobic channels, MGD-Chip enables the RPA and CRISPR-Cas12a assays to be sequentially performed on the device, ensuring minimal interference and cross-contamination. Six potential GD candidates were selected and successfully tested simultaneously on the platform within 1 h. Demonstrating exceptional specificity, the platform achieved a detection sensitivity of 0.1 nM for unamplified target plasmids and 1 aM for amplified ones. Validation using mouse models established by injecting IGFI and EPO transgenes confirmed the platform's efficacy in detecting gene doping in real samples. This technology, capable of detecting multiple targets using portable elements, holds promise for real-time GD detection at sports events, offering a rapid, highly sensitive, and user-friendly solution to uphold the integrity of sports competitions.
Subject(s)
Biosensing Techniques , CRISPR-Cas Systems , Doping in Sports , Hydrophobic and Hydrophilic Interactions , Lab-On-A-Chip Devices , CRISPR-Cas Systems/genetics , Doping in Sports/prevention & control , Animals , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , Mice , Humans , Erythropoietin/genetics , Erythropoietin/analysis , Equipment Design , CRISPR-Associated Proteins/genetics , Bacterial Proteins , EndodeoxyribonucleasesABSTRACT
The clinical diagnosis of pathogen infectious diseases increasingly requires sensitive and rapid RNA detection technologies. The RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system has shown immense potential in molecular diagnostics due to its trans-cleavage activity. However, most Cas13a-based detection methods require an amplicon transcription step, and the multi-step open-tube operations are prone to contamination, limiting their widespread application. Here, we propose an ultrasensitive (single-copy range, â¼aM) and rapid (within 40 min) isothermal one-pot RNA detection platform, termed SATCAS (Simultaneous Amplification and Testing platform based on Cas13a). This method effectively distinguishes viable bacteria (0%-100%) under constant total bacterial conditions, demonstrating its robustness and universality. SATCAS excels in identifying single nucleotide polymorphisms (SNPs), particularly detecting 0.5% drug-resistant mutations. We validated SATCAS by detecting infections in biological samples from 68 HBV, 23 EBV, and 48 SARS-CoV-2 patients, achieving 100% sensitivity, 92.86% specificity, and 97.06% accuracy in HBV infection testing. We anticipate that SATCAS has broad application potential in the early diagnosis, subtyping, drug resistance detection, and point-of-care monitoring of pathogen infectious diseases.
Subject(s)
Biosensing Techniques , CRISPR-Cas Systems , Nucleic Acid Amplification Techniques , Polymorphism, Single Nucleotide , SARS-CoV-2 , Humans , Biosensing Techniques/methods , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Nucleic Acid Amplification Techniques/methods , COVID-19/diagnosis , COVID-19/virology , RNA, Viral/genetics , Molecular Diagnostic Techniques/methods , Hepatitis B virus/genetics , Hepatitis B virus/isolation & purificationSubject(s)
Epigenesis, Genetic , Gene Editing , Humans , Gene Editing/methods , CRISPR-Cas Systems , Epigenomics/methods , AnimalsABSTRACT
During the repair of interstrand crosslinks (ICLs) a DNA double-strand break (DSB) is generated. The Fanconi anemia (FA) core complex, which is recruited to ICLs, promotes high-fidelity repair of this DSB by homologous recombination (HR). However, whether the FA core complex also promotes HR at ICL-independent DSBs, for example induced by ionizing irradiation or nucleases, remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen. Using isogenic cell line models, we further demonstrated an HR-promoting function of FANCL and Ube2T, and of their ubiquitination substrate FANCD2. We show that FANCL and Ube2T localize at DSBs in a FANCM-dependent manner, and are required for the DSB accumulation of FANCD2. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of CtIP at DSBs, thereby promoting end resection and Rad51 loading. Together, these data demonstrate a dual genome maintenance function of the FA core complex and FANCD2 in promoting repair of both ICLs and DSBs.
Subject(s)
DNA Breaks, Double-Stranded , Fanconi Anemia Complementation Group D2 Protein , Fanconi Anemia Complementation Group L Protein , Homologous Recombination , Ubiquitin-Conjugating Enzymes , Humans , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia Complementation Group D2 Protein/genetics , Fanconi Anemia Complementation Group L Protein/metabolism , Fanconi Anemia Complementation Group L Protein/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Carrier Proteins/metabolism , Carrier Proteins/genetics , CRISPR-Cas Systems , Ubiquitination , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Endodeoxyribonucleases/metabolism , Endodeoxyribonucleases/genetics , HEK293 Cells , Recombinational DNA Repair , DNA Repair , DNA End-Joining Repair , DNA HelicasesABSTRACT
In recent years, foodborne diseases have posed a serious threat to human health, and rapid detection of foodborne pathogens is particularly crucial for the prevention and control of such diseases. This article offers a detailed overview of the development of detection techniques for foodborne pathogens, transitioning from traditional microbiological culture methods to the current array of techniques, including immunological, molecular biological, and biosensor-based methods. It summarizes the technical principles, advantages, disadvantages, and research progress of these diverse methods. Furthermore, the article demonstrates that the combination of different methods enhances the efficiency and accuracy of pathogens detection. Specifically, the article focuses on the application and advantages of combining CRISPR/Cas systems with other detection methods in the detection of foodborne pathogens. CRISPR/Cas systems, with their high specificity, sensitivity, and ease of operation, show great potential in the field of foodborne pathogens detection. When integrated with other detection techniques such as immunological detection techniques, molecular biology detection techniques, and biosensors, the accuracy and efficiency of detection can be further improved. By fully utilizing these tools, early detection and control of foodborne diseases can be achieved, enhancing public health and preventing disease outbreaks. This article serves as a valuable reference for exploring more convenient, accurate, and sensitive field detection methods for foodborne pathogens, promoting the application of rapid detection techniques, and ensuring food safety and human health.
Subject(s)
Biosensing Techniques , Food Microbiology , Food Safety , Foodborne Diseases , Foodborne Diseases/microbiology , Foodborne Diseases/prevention & control , Food Microbiology/methods , Food Safety/methods , Humans , Biosensing Techniques/methods , CRISPR-Cas Systems , Food Contamination/analysisABSTRACT
Although significant progress has been made in the development of novel targeted drugs for the treatment of acute myeloid leukemia (AML) in recent years, chemotherapy still remains the mainstay of treatment and the overall survival is poor in most patients. Here, we demonstrated the antileukemia activity of a novel small molecular compound NL101, which is formed through the modification on bendamustine with a suberanilohydroxamic acid (SAHA) radical. NL101 suppresses the proliferation of myeloid malignancy cells and primary AML cells. It induces DNA damage and caspase 3-mediated apoptosis. A genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) library screen revealed that phosphatase and tensin homologous (PTEN) gene is critical for the regulation of cell survival upon NL101 treatment. The knockout or inhibition of PTEN significantly reduced NL101-induced apoptosis in AML and myelodysplastic syndrome (MDS) cells, accompanied by the activation of protein kinase B (AKT) signaling pathway. The inhibition of mammalian target of rapamycin (mTOR) by rapamycin enhanced the sensitivity of AML cells to NL101-induced cell death. These findings uncover PTEN protein expression as a major determinant of chemosensitivity to NL101 and provide a novel strategy to treat AML with the combination of NL101 and rapamycin.
Subject(s)
Apoptosis , Clustered Regularly Interspaced Short Palindromic Repeats , Leukemia, Myeloid, Acute , PTEN Phosphohydrolase , Humans , PTEN Phosphohydrolase/metabolism , PTEN Phosphohydrolase/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Survival/drug effects , Drug Resistance, Neoplasm , CRISPR-Cas SystemsABSTRACT
The coevolutionary dynamics of lytic viruses and microbes with CRISPR-Cas immunity exhibit alternations between sustained host control of viral proliferation and major viral epidemics in previous computational models. These alternating dynamics have yet to be observed in other host-pathogen systems. Here, we address the breakdown of control and transition to large outbreaks with a stochastic eco-evolutionary model. We establish the role of host density-dependent competition in punctuated virus-driven succession and associated diversity trends that concentrate escape pathways during control phases. Using infection and escape networks, we derive the viral emergence probability whose fluctuations of increasing size and frequency characterize the approach to large outbreaks. We explore alternation probabilities as a function of non-dimensional parameters related to the probability of viral escape and host competition. Our results demonstrate how emergent feedbacks between host competition and viral diversification render the host immune structure fragile, potentiating a dynamical transition to large epidemics.
Subject(s)
CRISPR-Cas Systems , Host-Pathogen Interactions/genetics , Models, Biological , Viruses/genetics , Evolution, Molecular , Biological EvolutionABSTRACT
We designed a simulation program that mimics the CRISPR-Cas9 editing on evolving barcode and double strand break repair procedure along with cell divisions. Emerging barcode mutations tend to build upon previously existing mutations, occurring sequentially with each generation. This process results in a unique mutation profile in each cell. We sample the barcodes in leaf cells and reconstruct the lineage, comparing it to the original lineage tree to test algorithm accuracy under different parameter settings. Our computational simulations validate the reasonable assumptions deduced from experimental observations, emphasizing that factors such as sampling size, barcode length, multiple barcodes, indel probabilities, and Cas9 activity are critical for accurate and successful lineage tracing. Among the many factors we found that sampling size and indel probabilities are two major ones that affect lineage tracing accuracy. Large segment deletions in early generations could greatly impact lineage accuracy. These simulation results offer insightful recommendations for enhancing the design and analysis of Cas9-mediated molecular barcodes in actual experiments.
Subject(s)
CRISPR-Cas Systems , Computer Simulation , Gene Editing , Gene Editing/methods , Algorithms , DNA Barcoding, Taxonomic/methods , Cell Lineage/genetics , INDEL Mutation , MutationABSTRACT
Therapeutic human gene editing technologies continue to advance, with the endonuclease, clustered regularly interspaced short palindromic repeats (CRISPR) being one of the most rapidly developing technologies. Recently, in 2024, a method of RNA editing called 'bridge editing' has been described in bacteria, which is more powerful and has broader applications than CRISPR to reshape the genome. The term 'bridge editing' is used because the method physically links, or bridges, two sections of DNA and can alter large sections of a genome. 'Bridge editing' relies on insertion sequence (IS) elements, the simplest autonomous transposable elements in prokaryotic genomes. This method provides a unified mechanism for the three fundamental types of DNA rearrangement required for genome design: inversion, insertion, and excision. The 'bridge' recombination system could expand the range and diversity of nucleic acid-guided therapeutic systems beyond RNA interference and CRISPR. This editorial aims to introduce new developments in 'bridge' RNA editing that have the increased potential to reshape the genome.
Subject(s)
CRISPR-Cas Systems , Gene Editing , RNA Editing , Gene Editing/methods , RNA Editing/genetics , Humans , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genome/genetics , DNA Transposable Elements/geneticsABSTRACT
Rectifying the aberrant microenvironment of a disease through maintenance of redox homeostasis has emerged as a promising perspective with significant therapeutic potential for Alzheimer's disease (AD). Herein, we design and construct a novel nanozyme-boosted MOF-CRISPR platform (CMOPKP), which can maintain redox homeostasis and rescue the impaired microenvironment of AD. By modifying the targeted peptides KLVFFAED, CMOPKP can traverse the blood-brain barrier and deliver the CRISPR activation system for precise activation of the Nrf2 signaling pathway and downstream redox proteins in regions characterized by oxidative stress, thereby reinstating neuronal antioxidant capacity and preserving redox homeostasis. Furthermore, cerium dioxide possessing catalase enzyme-like activity can synergistically alleviate oxidative stress. Further in vivo studies demonstrate that CMOPKP can effectively alleviate cognitive impairment in 3xTg-AD mouse models. Therefore, our design presents an effective way for regulating redox homeostasis in AD, which shows promise as a therapeutic strategy for mitigating oxidative stress in AD.
Subject(s)
Alzheimer Disease , Oxidative Stress , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Alzheimer Disease/genetics , Animals , Mice , Oxidative Stress/drug effects , Humans , NF-E2-Related Factor 2/metabolism , Metal-Organic Frameworks/chemistry , Disease Models, Animal , CRISPR-Cas Systems/genetics , Cerium/chemistry , Cerium/therapeutic use , Cerium/pharmacology , Blood-Brain Barrier/metabolism , Oxidation-Reduction , Antioxidants/chemistry , Antioxidants/pharmacology , Antioxidants/therapeutic useABSTRACT
Resistance to radiotherapy is a major barrier during cancer treatment. Here using genome-scale CRISPR/Cas9 screening, we identify CD274 gene, which encodes PD-L1, to confer lung cancer cell resistance to ionizing radiation (IR). Depletion of endogenous PD-L1 delays the repair of IR-induced DNA double-strand breaks (DSBs) and PD-L1 loss downregulates non-homologous end joining (NHEJ) while overexpression of PD-L1 upregulates NHEJ. IR induces translocation of PD-L1 from the membrane into nucleus dependent on deglycosylation of PD-L1 at N219 and CMTM6 and leads to PD-L1 recruitment to DSBs foci. PD-L1 interacts with Ku in the nucleus and enhances Ku binding to DSB DNA. The interaction between the IgC domain of PD-L1 and the core domain of Ku is required for PD-L1 to accelerate NHEJ-mediated DSB repair and produce radioresistance. Thus, PD-L1, in addition to its immune inhibitory activity, acts as mechanistic driver for NHEJ-mediated DSB repair in cancer.
Subject(s)
B7-H1 Antigen , Cell Nucleus , DNA Breaks, Double-Stranded , DNA End-Joining Repair , Ku Autoantigen , Humans , DNA Breaks, Double-Stranded/radiation effects , B7-H1 Antigen/metabolism , B7-H1 Antigen/genetics , Ku Autoantigen/metabolism , Ku Autoantigen/genetics , Cell Line, Tumor , Cell Nucleus/metabolism , Animals , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/radiotherapy , Lung Neoplasms/pathology , Mice , Glycosylation , Radiation, Ionizing , CRISPR-Cas SystemsABSTRACT
BACKGROUND: CRISPR-Cas13a is renowned for its precise and potent RNA editing capabilities in cancer therapy. While various material systems have demonstrated efficacy in supporting CRISPR-Cas13a to execute cellular functions in vitro efficiently and specifically, the development of CRISPR-Cas13a-based therapeutic agents for intravesical instillation in bladder cancer (BCa) remains unexplored. METHODS: In this study, we introduce a CRISPR-Cas13a nanoplatform, which effectively inhibits PDL1 expression following intravesical instillation. This system utilizes a fusion protein CAST, created through the genetic fusion of CRISPR-Cas13 and the transmembrane peptide TAT. CAST acts as a potent transmembrane RNA editor and is assembled with the transepithelial delivery carrier fluorinated chitosan (FCS). Upon intravesical administration into the bladder, the CAST-crRNAa/FCS nanoparticles (NPs) exhibit remarkable transepithelial capabilities, significantly suppressing PDL1 expression in tumor tissues.To augment immune activation within the tumor microenvironment, we integrated a fenbendazole (FBZ) intravesical system (FBZ@BSA/FCS NPs). This system is formulated through BSA encapsulation followed by FCS coating, positioning FBZ as a powerful chemo-immunological agent. RESULTS: In an orthotropic BCa model, the FBZ@BSA/FCS NPs demonstrated pronounced tumor cell apoptosis, synergistically reduced PDL1 expression, and restructured the immune microenvironment. This culminated in an enhanced synergistic intravesical instillation approach for BCa. Consequently, our study unveils a novel RNA editor nanoagent formulation and proposes a potential synergistic therapeutic strategy. This approach significantly bolsters therapeutic efficacy, holding promise for the clinical translation of CRISPR-Cas13-based cancer perfusion treatments.
Subject(s)
CRISPR-Cas Systems , Urinary Bladder Neoplasms , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/therapy , Humans , Animals , Administration, Intravesical , Mice , Cell Line, Tumor , FemaleABSTRACT
Function as a potential cancer biomarker, DNA methylation shows great significance in cancer diagnosis, prognosis, and treatment monitoring. While the lack of an ultrasensitive, specific, and accurate method at the single-molecule level hinders the analysis of the exceedingly low levels of DNA methylation. Herein, based on the outstanding recognition and digestion ability of methylation-sensitive restriction endonuclease (MSRE), we established a single MSRE-based cascade exponential amplification method, which requires only two ingeniously designed primers and only one recognition site of MSRE for the detection of DNA methylation. Differentiated by MSRE digestion, the cleaved unmethylated DNA is too short to induce any amplification reactions, while methylated DNA remains intact to trigger cascade exponential amplification and the subsequent CRISPR/Cas12a system. By integrating the two exponential amplification reactions, as low as 1 aM methylated DNA can be accurately detected, which corresponds to 6 molecules in a 10 µL system, indicating that our method is more sensitive than single amplification-based methods with the ability to detect DNA methylation at the single-molecule level. In addition, 0.1% methylated DNA can be effectively distinguished from large amounts of unmethylated DNA. Our method is further introduced to exploit the expression difference of DNA methylation among normal cells and cancer cells. Moreover, the visual detection of DNA methylation is also realized by the full hybridization between amplification products and the crRNA of CRISPR/Cas12a. Therefore, the proposed method has great potential to be a promising and robust bisulfite-free method for the detection of DNA methylation at the single-molecule level, which is of great importance for early diagnosis of cancer.
Subject(s)
DNA Methylation , DNA Restriction Enzymes , Nucleic Acid Amplification Techniques , Humans , Nucleic Acid Amplification Techniques/methods , DNA Restriction Enzymes/metabolism , CRISPR-Cas Systems/genetics , DNA/chemistry , DNA/geneticsABSTRACT
Abnormal expression in long noncoding RNAs (lncRNAs) is closely associated with cancers. Herein, a novel CRISPR/Cas13a-enhanced photocurrent-polarity-switching photoelectrochemical (PEC) biosensor was engineered for the joint detection of dual lncRNAs, using deep learning (DL) to assist in cancer diagnosis. After target lncRNA-activated CRISPR/Cas13a cleaves to induce DNAzyme bidirectional walkers with the help of cofactor Mg2+, nitrogen-doped carbon-Cu/Cu2O octahedra are introduced into the biosensor, producing a photocurrent in the opposite direction of CdS quantum dots (QDs). The developed PEC biosensor shows high specificity and sensitivity with limits of detection down to 25.5 aM for lncRNA HOTAIR and 53.1 aM for lncRNA MALAT1. More importantly, this platform for the lncRNA joint assay in whole blood can successfully differentiate cancers from healthy people. Furthermore, the DL model is applied to explore the potential pattern hidden in data of the established technology, and the accuracy of DL cancer diagnosis can acquire 93.3%. Consequently, the developed platform offers a new avenue for lncRNA joint detection and early intelligent diagnosis of cancer.