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Gene therapy using a protein-based CRISPR system in the brain has practical limitations due to current delivery systems, especially in the presence of arterial occlusion. To overcome these obstacles and improve stability, we designed a system for intranasal administration of gene therapy for the treatment of ischemic stroke. Methods: Nanoparticles containing the protein-based CRISPR/dCas9 system targeting Sirt1 were delivered intranasally to the brain in a mouse model of ischemic stroke. The CRISPR/dCas9 system was encapsulated with calcium phosphate (CaP) nanoparticles to prevent them from being degraded. They were then conjugated with ß-hydroxybutyrates (bHb) to target monocarboxylic acid transporter 1 (MCT1) in nasal epithelial cells to facilitate their transfer into the brain. Results: Human nasal epithelial cells were shown to uptake and transfer nanoparticles to human brain endothelial cells with high efficiency in vitro. The intranasal administration of the dCas9/CaP/PEI-PEG-bHb nanoparticles in mice effectively upregulated the target gene, Sirt1, in the brain, decreased cerebral edema and increased survival after permanent middle cerebral artery occlusion. Additionally, we observed no significant in vivo toxicity associated with intranasal administration of the nanoparticles, highlighting the safety of this approach. Conclusion: This study demonstrates that the proposed protein-based CRISPR-dCas9 system targeting neuroprotective genes in general, and SIRT1 in particular, can be a potential novel therapy for acute ischemic stroke.
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Administración Intranasal , Encéfalo , Modelos Animales de Enfermedad , Terapia Genética , Accidente Cerebrovascular Isquémico , Nanopartículas , Sirtuina 1 , Animales , Ratones , Humanos , Accidente Cerebrovascular Isquémico/terapia , Accidente Cerebrovascular Isquémico/genética , Nanopartículas/administración & dosificación , Terapia Genética/métodos , Sirtuina 1/genética , Sirtuina 1/metabolismo , Encéfalo/metabolismo , Masculino , Fosfatos de Calcio , Sistemas CRISPR-Cas , Ratones Endogámicos C57BL , Células Endoteliales/metabolismo , Isquemia Encefálica/terapia , Isquemia Encefálica/genética , Infarto de la Arteria Cerebral Media/terapia , Infarto de la Arteria Cerebral Media/genética , Células Epiteliales/metabolismoRESUMEN
Understanding the spatial organization of genomes within chromatin is crucial for deciphering gene regulation. A recently developed CRISPR-dCas9-based genome labeling tool, known as CRISPR-FISH, allows efficient labelling of repetitive sequences. Unlike standard fluorescence in situ hybridization (FISH), CRISPR-FISH eliminates the need for global DNA denaturation, allowing for superior preservation of chromatin structure. Here, we report on the further development of the CRISPR-FISH method, which has been enhanced for increased efficiency through the engineering of a recombinant dCas9 protein containing an ALFA-tag. Using an ALFA-tagged dCas9 protein assembled with an A. thaliana centromere-specific gRNA, we demonstrate target-specific labelling with a fluorescence-labeled NbALFA nanobody. The dCas9 protein possessing multiple copies of the ALFA-tag, in combination with a minibody and fluorescence-labelled anti-rabbit secondary antibody, resulted in enhanced target-specific signals. The dCas9-ALFA-tag system was also instrumental in live cell imaging of telomeres in N. benthamiana. This method will further expand the CRISPR imaging toolkit, facilitating a better understanding of genome organization. Furthermore, we report the successful integration of the highly sensitive Tyramide Signal Amplification (TSA) method with CRISPR-FISH, demonstrating effective labeling of A. thaliana centromeres.
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The utilization of Streptomyces as a microbial chassis for developing innovative drugs and medicinal compounds showcases its capability to produce bioactive natural substances. Recent focus on the clustered regularly interspaced short palindromic repeat (CRISPR) technology highlights its potential in genome editing. However, applying CRISPR technology in certain microbial strains, particularly Streptomyces, encounters specific challenges. These challenges include achieving efficient gene expression and maintaining genetic stability, which are critical for successful genome editing. To overcome these obstacles, an innovative approach has been developed that combines several key elements: activation-induced cytidine deaminase (AID), nuclease-deficient cas9 variants (dCas9), and Petromyzon marinus cytidine deaminase 1 (PmCDA1). In this study, this novel strategy was employed to engineer a Streptomyces coelicolor strain. The target gene was actVA-ORF4 (SCO5079), which is involved in actinorhodin production. The engineering process involved introducing a specific construct [pGM1190-dcas9-pmCDA-UGI-AAV-actVA-ORF4 (SCO5079)] to create a CrA10 mutant strain. The resulting CrA10 mutant strain did not produce actinorhodin. This outcome highlights the potential of this combined approach in the genetic manipulation of Streptomyces. The failure of the CrA10 mutant to produce actinorhodin conclusively demonstrates the success of gene editing at the targeted site, affirming the effectiveness of this method for precise genetic modifications in Streptomyces.
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The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system, a groundbreaking innovation in genetic engineering, has revolutionized our approach to surmounting complex diseases, culminating in CASGEVY™ approved for sickle cell anemia. Derived from a microbial immune defense mechanism, CRISPR/Cas9, characterized as precision, maneuverability and universality in gene editing, has been harnessed as a versatile tool for precisely manipulating DNA in mammals. In the process of applying it to practice, the consecutive exploitation of novel orthologs and variants never ceases. It's conducive to understanding the essentialities of diseases, particularly cancer, which is crucial for diagnosis, prevention, and treatment. CRISPR/Cas9 is used not only to investigate tumorous genes functioning but also to model disparate cancers, providing valuable insights into tumor biology, resistance, and immune evasion. Upon cancer therapy, CRISPR/Cas9 is instrumental in developing individual and precise cancer therapies that can selectively activate or deactivate genes within tumor cells, aiming to cripple tumor growth and invasion and sensitize cancer cells to treatments. Furthermore, it facilitates the development of innovative treatments, enhancing the targeting efficiency of reprogrammed immune cells, exemplified by advancements in CAR-T regimen. Beyond therapy, it is a potent tool for screening susceptible genes, offering the possibility of intervening before the tumor initiative or progresses. However, despite its vast potential, the application of CRISPR/Cas9 in cancer research and therapy is accompanied by significant efficacy, efficiency, technical, and safety considerations. Escalating technology innovations are warranted to address these issues. The CRISPR/Cas9 system is revolutionizing cancer research and treatment, opening up new avenues for advancements in our understanding and management of cancers. The integration of this evolving technology into clinical practice promises a new era of precision oncology, with targeted, personalized, and potentially curative therapies for cancer patients.
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Sistemas CRISPR-Cas , Neoplasias , Medicina de Precisión , Humanos , Sistemas CRISPR-Cas/genética , Medicina de Precisión/métodos , Neoplasias/genética , Neoplasias/terapia , Edición Génica/métodos , Animales , Oncología Médica/métodos , Oncología Médica/tendenciasRESUMEN
The dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi) gene regulation technique requires two components: a catalytically inactive Cas9 protein (dCas9) and a single-guide RNA that targets the gene of interest. This system is commonly activated by expressing dCas9 through an inducible gene promoter, but these inducers may affect cellular physiology, and accessibility and permeability of the inducer are limited in relevant model systems. Here, we have developed an alternative approach for CRISPRi activation in the clinical isolate Staphylococcus aureus USA300 LAC, where dCas9 was expressed through endogenous virulence gene promoters (vgp); coagulase, autolysin, or fibronectin-binding protein A. Additionally, we integrated a fluorescent reporter gene into the vgp-CRISPRi system to monitor the activity of the dcas9-controlling promoter. Testing the efficacy of vgp-CRISPRi by inducing growth arrest (when targeting penicillin-binding protein 1), downregulating target gene expression, or blocking coagulase-dependent coagulation of blood plasma, we provide a proof-of-concept demonstration that the virulence gene promoter-driven CRISPRi system is functional in S. aureus.IMPORTANCEThe presented inducer-free, endogenous virulence gene promoter-induced, dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi system addresses several shortcomings related to the use of inducer-dependent systems such as effects on cell physiology or limitations in permeability, and it avoids the high, putatively toxic levels of dCas9 in CRISPRi systems controlled by strong, constitutive promoters.
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Although therapeutic targets for colorectal cancer (CRC) treatment have been developed, the treatment outcomes are not ideal and survival rates for CRC patients remain low. It is critical to identify a specific target and develop an effective CRC treatment system. The ZNF334 gene is a newly identified member of Zinc-finger proteins (ZNFs), which is essential for key biological processes associated with tumorigenesis. Abnormal epigenetic reprogramming of the ZNF334 gene promoter region decreases its expression in CRC and further induces the occurrence of CRC. Here, we clarified that P300 in CRC can regulate the H3K9/27 ac in the ZNF334 promoter. Furthermore, histone acetylation of the ZNF334 promoter region was increased by dCas9-P300 to normalize the deficiency of ZNF334 expression, thereby inhibiting the growth of CRC. Collectively, our findings enable a facile way to affect gene expression using CRISPR/Cas9-based epigenome editing and further determine the causal link between histone acetylation and gene activation, providing a promising gene therapy strategy for the CRC treatment.
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Sistemas CRISPR-Cas , Neoplasias Colorrectales , Epigénesis Genética , Edición Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Edición Génica/métodos , Acetilación , Proliferación Celular/genética , Regiones Promotoras Genéticas/genética , Animales , Línea Celular Tumoral , Histonas/metabolismo , Ratones , Acetiltransferasas/genética , Acetiltransferasas/metabolismoRESUMEN
Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disease. Although it leads to muscle weakness, affected individuals predominantly die from cardiomyopathy, which remains uncurable. Accumulating evidence suggests that an overexpression of utrophin may counteract some of the pathophysiological outcomes of DMD. The aim of this study was to investigate the role of utrophin in dystrophin-deficient human cardiomyocytes (CMs) and to test whether an overexpression of utrophin, implemented via the CRISPR-deadCas9-VP64 system, can improve their phenotype. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) lacking either dystrophin (DMD) or both dystrophin and utrophin (DMD KO/UTRN(+/-)). We carried out proteome analysis, which revealed considerable differences in the proteins related to muscle contraction, cell-cell adhesion, and extracellular matrix organization. Furthermore, we evaluated the role of utrophin in maintaining the physiological properties of DMD hiPSC-CMs using atomic force microscopy, patch-clamp, and Ca2+ oscillation analysis. Our results showed higher values of afterhyperpolarization and altered patterns of cytosolic Ca2+ oscillations in DMD; the latter was further disturbed in DMD KO/UTRN(+/-) hiPSC-CMs. Utrophin upregulation improved both parameters. Our findings demonstrate for the first time that utrophin maintains the physiological functions of DMD hiPSC-CMs, and that its upregulation can compensate for the loss of dystrophin.
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DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are considered worthwhile and many epigenome editing technologies have been developed, which were based on ZF, TALE, and CRISPR-dCas9. Here, we describe a protocol for the application of a modified dCas9-SunTag system, which increased the efficiency of targeted demethylation and gene activation at specific DNA loci. The original SunTag system consists of 10 copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve more efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, an enzyme that demethylates DNA, we changed the linker length to 22 amino acids. Moreover, we describe the co-recruitment of TET1 and VP64 for efficient gene activation. Since we showed the manipulation of DNA methylation at specific loci and gene activation, its application could lead to its future use in the clinic.
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Sistemas CRISPR-Cas , Metilación de ADN , Humanos , Edición Génica/métodos , Regulación de la Expresión Génica , Epigénesis Genética , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genéticaRESUMEN
Epigenetic editing enables the locus-specific manipulation of chromatin modifications. It allows the functional analysis of interactions between chromatin modifications and epigenetically stable gene expression states, thus establishing causal relationships, where previously correlations were suspected. Here, we describe the procedures for gene-specific epigenetic editing in plants that are based on targeting a histone modifier using an inactive dCas9 fusion protein that is recruited by a set of three distinct single guide RNAs (sgRNAs) that all target a region within the promoter of the target gene. We outline design principles and emphasize the need for suitable control constructs. In summary, the protocol will be widely useful for plant scientists looking to manipulate chromatin modifications in a locus-specific manner.
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Epigénesis Genética , Edición Génica , Regulación de la Expresión Génica de las Plantas , Histonas , Edición Génica/métodos , Histonas/metabolismo , Histonas/genética , ARN Guía de Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas , Código de Histonas , Cromatina/genética , Cromatina/metabolismo , Regiones Promotoras Genéticas , Plantas Modificadas Genéticamente/genética , Plantas/genética , Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismoRESUMEN
In this chapter, we present an experimental protocol to conduct DNA methylation editing experiments, that is, to induce loss or gain of DNA methylation, targeting Dlk1-Dio3 imprinted domain, a well-studied imprinted locus, in ES cells. In this protocol, plasmid vectors expressing the DNA methylation editing tools, combining the CRISPR/dCas9 system and the SunTag system coupled to a DNA methyltransferase or a TET enzyme, are introduced into cells for transient expression. By employing this strategy, researchers can effectively investigate a distinct DNA methylation signature that has an impact on the imprinting status, including gene expression and histone modifications, across the entire domain. We also describe strategies for allele-specific quantitative analyses of DNA methylation, gene expression, and histone modifications and binding protein levels for assessing the imprinting state of the locus.
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Sistemas CRISPR-Cas , Metilación de ADN , Edición Génica , Impresión Genómica , Edición Génica/métodos , Animales , Ratones , Sitios Genéticos , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Yoduro Peroxidasa/genética , Alelos , HumanosRESUMEN
The discovery and adaptation of CRISPR/Cas systema for epigenome editing has allowed for a straightforward design of targeting modules that can direct epigenome editors to virtually any genomic site. This advancement in DNA-targeting technology brings allele-specific epigenome editing into reach, a "super-specific" variation of epigenome editing whose goal is an alteration of chromatin marks at only one selected allele of the genomic target locus. This technology could be useful for the treatment of diseases caused by a mutant allele with a dominant effect, because allele-specific epigenome editing allows the specific silencing of the mutated allele leaving the healthy counterpart expressed. Moreover, it may allow the direct correction of aberrant imprints in imprinting disorders where editing of DNA methylation is required exclusively in a single allele. Here, we describe a basic protocol for the design and application of allele-specific epigenome editing systems using allele-specific DNA methylation at the NARF gene in HEK293 cells as an example. An sgRNA/dCas9 unit is used for allele-specific binding to the target locus containing a SNP in the seed region of the sgRNA or the PAM region. The dCas9 protein is connected to a SunTag allowing to recruit up to 10 DNMT3A/3L units fused to a single-chain Fv fragment, which specifically binds to the SunTag peptide sequence. The plasmids expressing dCas9-10x SunTag, scFv-DNMT3A/3L, and sgRNA, each of them co-expressing a fluorophore, are introduced into cells by co-transfection. Cells containing all three plasmids are enriched by FACS, cultivated, and later the genomic DNA and RNA can be retrieved for DNA methylation and gene expression analysis.
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Alelos , Sistemas CRISPR-Cas , Metilación de ADN , Epigenoma , Edición Génica , Humanos , Edición Génica/métodos , Células HEK293 , ARN Guía de Sistemas CRISPR-Cas/genética , Epigenómica/métodos , Epigénesis GenéticaRESUMEN
Genome editing tools, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (e.g., CRISPR/Cas9), and their repurposing into epigenetic editing platforms, offer enormous potential as safe and customizable therapies for cancer. Specifically, various transcriptional abnormalities in human malignancies, such as silencing of tumor suppressors and ectopic re-expression of oncogenes, have been successfully targeted with virtually no off-target effects using CRISPR activation and repression systems. In these systems, the nuclease-deactivated Cas9 protein (dCas9) is fused to one or more domains inducing selective activation or repression of the targeted genes. Despite these advances, the efficient in vivo delivery of these molecules into the target cancer cells represents a critical barrier to accomplishing translation into a clinical therapy setting for cancer. Major obstacles include the large size of dCas9 fusion proteins, the necessity of multimodal delivery of protein and gRNAs, and the potential of these formulations to elicit detrimental immune responses.In this context, viral methods for delivering CRISPR face several limitations, such as the packaging capacity of the viral genome, the potential for integration of the nucleic acids into the host cells genome, and immunogenicity of viral proteins, posing serious safety concerns. The rapid development of mRNA vaccines in response to the COVID-19 pandemic has rekindled interest in mRNA-based approaches for CRISPR/dCas9 delivery. Simultaneously, due to their high loading capacity, scalability, customizable surface modification for cell targeting, and low immunogenicity, lipid nanoparticles (LNPs) have been widely explored as nonviral vectors. In this chapter, we first describe the design of optimized dCas9-effector mRNAs and gRNAs for epigenetic editing. We outline formulations of LNPs suitable for dCas9 mRNA delivery. Additionally, we provide a protocol for the co-encapsulation of the dCas9-effector mRNAs and gRNA into these LNPs, along with detailed methods for delivering these formulations to both cell lines (in vitro) and mouse models of breast cancer (in vivo).
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Sistemas CRISPR-Cas , Edición Génica , Nanopartículas , Neoplasias , Edición Génica/métodos , Humanos , Nanopartículas/química , Animales , Neoplasias/genética , Neoplasias/terapia , Epigénesis Genética , Ratones , ARN Guía de Sistemas CRISPR-Cas/genética , Liposomas/química , Línea Celular Tumoral , Lípidos/química , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Terapia Genética/métodos , Técnicas de Transferencia de GenRESUMEN
Epigenetic modifications play a crucial role in regulating gene expression patterns. Through epigenetic editing approaches, the chromatin structure is modified and the activity of the targeted gene can be reprogrammed without altering the DNA sequence. By using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic repeats) platform with nuclease-deactivated dCas9 proteins to direct epigenetic effector domains (EDs) to genomic regulatory regions, the expression of the targeted gene can be modulated. However, the long-term stability of these effects, although demonstrated, remains unpredictable. The versatility and flexibility of (co-)targeting different genes with multiple epigenetic effectors has made the CRISPR/dCas9 platform the most widely used gene modulating technology currently available. Efficient delivery of large dCas9-ED fusion constructs into target cells, however, is challenging. An approach to overcome this limitation is to generate cells that stably express sgRNA(s) or dCas9-ED constructs. The sgRNA(s) or dCas9-ED stable cell lines can be used to study the mechanisms underlying sustained gene expression reprogramming by transiently expressing the other of the two constructs. Here, we describe a detailed protocol for the engineering of cells that stably express CRISPR/dCas9 or sgRNA. Creating a system where one component of the CRISPR/dCas9 is stably expressed while the other is transiently expressed offers a versatile platform for investigating the dynamics of epigenetic reprogramming.
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Sistemas CRISPR-Cas , Epigénesis Genética , Edición Génica , ARN Guía de Sistemas CRISPR-Cas , Edición Génica/métodos , Humanos , ARN Guía de Sistemas CRISPR-Cas/genética , Línea Celular , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Células HEK293RESUMEN
To fully exploit the potentials of reprogramming the epigenome through CRISPR/dCas9 systems for epigenetic editing, there is a growing need for improved transfection methods. With the utilization of constructs often with large sizes and the wide array of cell types used to read out the effect of epigenetic editing in different biological applications, it is evident that ongoing optimalization of transfection protocols tailored to each specific experimental setup is essential. Whether the goal is the production of viral particles using human embryonic kidney (HEK) cells or the direct examination of epigenomic modifications in the target cell type, continuous refinement of transfection methods is crucial. In the hereafter outlined protocol, we focus on optimization of transfection protocols by comparing different reagents and methods, creating a streamlined setup for transfection efficiency optimization in cultured mammalian cells. Our protocol provides a comprehensive overview of flow cytometry analysis following transfection not just to improve transfection efficiency but also to assess the expression level of the utilized construct. We showcase our transfection protocol optimization using HEK293T Lenti-X™ and breast cancer MCF-7 cell lines, using a single-guide RNA-containing plasmid. Specifically, we incorporate heat shock treatment for increased transfection efficiency of the MCF-7 cell line. Our detailed optimization protocol for efficient plasmid delivery and measurement of single-cell plasmid expression provides a comprehensive instruction for assessing both transient and sustained effects of epigenetic reprogramming.
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Sistemas CRISPR-Cas , Epigénesis Genética , Edición Génica , Plásmidos , Análisis de la Célula Individual , Transfección , Humanos , Plásmidos/genética , Edición Génica/métodos , Células HEK293 , Transfección/métodos , Análisis de la Célula Individual/métodos , Epigenómica/métodos , Citometría de FlujoRESUMEN
DNA methylation is a key epigenetic mechanism orchestrating gene expression networks in many biological processes. Nonetheless, studying the role of specific gene methylation events in fish faces challenges. In this study, we validate the regulation of DNA methylation on empty spiracles homeobox 2 (emx2) expression with decitabine treatment in Chinese tongue sole testis cells. We used the emx2 gene as the target gene and developed a new DNA methylation editing system by fusing dnmt3a with catalytic dead Cas9 (dCas9) and demonstrated its ability for sequence-specific DNA methylation editing. Results revealed that utilizing dCas9-dnmt3a to target emx2 promoter region led to increased DNA methylation levels and decreased emx2 expression in Chinese tongue sole testis cells. More importantly, the DNA methylation editing significantly suppressed the expression of MYC proto-oncogene, bHLH transcription factor (myc), one target gene of emx2. Furthermore, we assessed the off-target effects of dCas9-dnmt3a and confirmed no significant impact on the predicted off-target gene expression. Taken together, we developed the first DNA methylation editing system in marine species and demonstrated its effective editing ability in Chinese tongue sole cells. This provides a new strategy for both epigenetic research and molecular breeding of marine species.
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Metilación de ADN , Edición Génica , Proteínas de Homeodominio , Testículo , Animales , Masculino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Testículo/metabolismo , Edición Génica/métodos , Sistemas CRISPR-Cas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Peces Planos/genética , Regiones Promotoras Genéticas/genética , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , ADN Metiltransferasa 3ARESUMEN
Epigenome editing has emerged as a powerful technique for targeted manipulation of the chromatin and transcriptional landscape, employing designer DNA binding domains fused with effector domains, known as epi-editors. However, the constitutive expression of dCas9-based epi-editors presents challenges, including off-target activity and lack of temporal resolution. Recent advancements of dCas9-based epi-editors have addressed these limitations by introducing innovative switch systems that enable temporal control of their activity. These systems allow precise modulation of gene expression over time and offer a means to deactivate epi-editors, thereby reducing off-target effects associated with prolonged expression. The development of novel dCas9 effectors regulated by exogenous chemical signals has revolutionized temporal control in epigenome editing, significantly expanding the researcher's toolbox. Here, we provide a comprehensive review of the current state of these cutting-edge systems and specifically discuss their advantages and limitations, offering context to better understand their capabilities.
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Epigénesis Genética , Edición Génica , Edición Génica/métodos , Humanos , Epigénesis Genética/efectos de los fármacos , Epigenoma , Sistemas CRISPR-Cas , Cromatina/genética , Cromatina/metabolismo , Epigenómica/métodos , AnimalesRESUMEN
Histone citrullination, an important post-translational modification mediated by peptidyl arginine deiminases, is essential for many physiological processes and epigenetic regulation. However, the causal relationship between histone citrullination and specific gene regulation remains unresolved. In this study, we develop a programmable epigenetic editor by fusing the peptidyl arginine deiminase PPAD from Porphyromonas gingivalis with dCas9. With the assistance of gRNA, PPAD-dCas9 can recruit peptidyl arginine deiminases to specific genomic loci, enabling direct manipulation of the epigenetic landscape and regulation of gene expression. Our citrullination editor allows for site-specific manipulation of histone H3R2,8,17 and 26 at target human gene loci, resulting in the activation or suppression of different genes in a locus-specific manner. Moreover, the epigenetic effects of the citrullination editor are specific and sustained. This epigenetic editor offers an accurate and efficient tool for exploring gene regulation of histone citrullination.
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Osteoarthritis (OA) is a painful and debilitating disease affecting over 500 million people worldwide. Intraarticular injection of mesenchymal stromal cells (MSCs) shows promise for the clinical treatment of OA, but the lack of consistency in MSC preparation and application makes it difficult to further optimize MSC therapy and to properly evaluate the clinical outcomes. In this study, we used Sox9 activation and RelA inhibition, both mediated by the CRISPR-dCas9 technology simultaneously, to engineer MSCs with enhanced chondrogenic potential and downregulated inflammatory responses. We found that both Sox9 and RelA could be fine-tuned to the desired levels, which enhances the chondrogenic and immunomodulatory potentials of the cells. Intraarticular injection of modified cells significantly attenuated cartilage degradation and palliated OA pain compared with the injection of cell culture medium or unmodified cells. Mechanistically, the modified cells promoted the expression of factors beneficial to cartilage integrity, inhibited the production of catabolic enzymes in osteoarthritic joints, and suppressed immune cells. Interestingly, a substantial number of modified cells could survive in the cartilaginous tissues including articular cartilage and meniscus. Together, our results suggest that CRISPR-dCas9-based gene regulation is useful for optimizing MSC therapy for OA.
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Sistemas CRISPR-Cas , Células Madre Mesenquimatosas , Osteoartritis , Factor de Transcripción SOX9 , Factor de Transcripción ReIA , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Osteoartritis/terapia , Osteoartritis/genética , Osteoartritis/metabolismo , Células Madre Mesenquimatosas/metabolismo , Animales , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción ReIA/genética , Ratones , Humanos , Modelos Animales de Enfermedad , Cartílago Articular/metabolismo , Cartílago Articular/patología , Trasplante de Células Madre Mesenquimatosas/métodos , Condrogénesis/genética , Edición Génica , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Condrocitos/metabolismoRESUMEN
Cancer cells experiencing hypoxic stress employ epithelial-mesenchymal transition (EMT) to undergo metastasis through rewiring of the chromatin landscape, epigenetics, and importantly, gene expression. Here, we showed that hypoxia modulates the epigenetic landscape on CTCF promoter and upregulates its expression. Hypoxia-driven epigenetic regulation, specifically DNA demethylation mediated by TET2, is a prerequisite for CTCF induction. Mechanistically, in hypoxic conditions, Hypoxia-inducible factor 1-alpha (HIF1α) binds to the unmethylated CTCF promoter, causing transcriptional upregulation. Further, we uncover the pivotal role of CTCF in promoting EMT as loss of CTCF abrogated invasiveness of hypoxic breast cancer cells. These findings highlight the functional contribution of HIF1α-CTCF axis in promoting EMT in hypoxic breast cancer cells. Lastly, CTCF expression is alleviated and the potential for EMT is diminished when the HIF1α binding is particularly disrupted through the dCas9-DNMT3A system-mediated maintenance of DNA methylation on the CTCF promoter. This axis may offer a unique therapeutic target in breast cancer.
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Neoplasias de la Mama , Factor de Unión a CCCTC , Hipoxia de la Célula , Metilación de ADN , Transición Epitelial-Mesenquimal , Subunidad alfa del Factor 1 Inducible por Hipoxia , Regiones Promotoras Genéticas , Humanos , Factor de Unión a CCCTC/metabolismo , Transición Epitelial-Mesenquimal/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Femenino , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Regiones Promotoras Genéticas/genética , Metilación de ADN/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Dioxigenasas , Epigénesis Genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ADN Metiltransferasa 3A/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Epstein-Barr virus (EBV) is a ubiquitous human tumor virus that establishes lifelong, persistent infections in B cells. The presence of EBV in cancer cells presents an opportunity to target these cells by reactivating the virus from latency. In this study, we developed a novel approach for EBV reactivation termed clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated EBV reactivation (CMER) strategy. Using modified CRISPR-associated protein 9 (dCas9) fused with VP64, we designed 10 single guide RNAs (sgRNAs) to target and activate the EBV immediate-early gene promoter. In Akata Burkitt lymphoma cells, 9 out of 10 CMER sgRNAs effectively reactivated EBV. Among these, CMER sgRNA-5 triggered robust reactivation across various cell types, including lymphoma, gastric cancer, and nasopharyngeal carcinoma cells. Importantly, the combination of CMER and ganciclovir selectively eliminated EBV-positive cells, regardless of their cell origin. These findings indicate that targeted virus reactivation by CMER, combined with nucleoside analog therapy, holds promise for EBV-associated cancer treatment. IMPORTANCE: This study explores a novel strategy called clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated Epstein-Barr virus (EBV) reactivation (CMER) to reactivate the Epstein-Barr virus in cancer cells. EBV is associated with various cancers, and reactivating EBV from latency offers a potential therapeutic strategy. We utilized an enzymatically inactive CRISPR-associated protein 9 (dCas9) fused with VP64 and designed 10 single guide RNAs to target the EBV immediate-early gene promoter. Nine of these sgRNAs effectively reactivated EBV in Burkitt lymphoma cells, with CMER sgRNA-5 demonstrating strong reactivation across different cancer cell types. Combining CMER with ganciclovir selectively eliminated EBV-positive cells, showing promise for EBV-associated cancer treatment.