RESUMO
CRISPR activation (CRISPRa) is an important tool to perturb transcription, but its effectiveness varies between target genes. We employ human pluripotent stem cells with thousands of randomly integrated barcoded reporters to assess epigenetic features that influence CRISPRa efficacy. Basal expression levels are influenced by genomic context and dramatically change during differentiation to neurons. Gene activation by dCas9-VPR is successful in most genomic contexts, including developmentally repressed regions, and activation level is anti-correlated with basal gene expression, whereas dCas9-p300 is ineffective in stem cells. Certain chromatin states, such as bivalent chromatin, are particularly sensitive to dCas9-VPR, whereas constitutive heterochromatin is less responsive. We validate these rules at endogenous genes and show that activation of certain genes elicits a change in the stem cell transcriptome, sometimes showing features of differentiated cells. Our data provide rules to predict CRISPRa outcome and highlight its utility to screen for factors driving stem cell differentiation.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas CRISPR-Cas , Neurônios , Ativação Transcricional , Cromatina/genéticaRESUMO
Despite extensive studies on the chromatin landscape of exhausted T cells, the transcriptional wiring underlying the heterogeneous functional and dysfunctional states of human tumor-infiltrating lymphocytes (TILs) is incompletely understood. Here, we identify gene-regulatory landscapes in a wide breadth of functional and dysfunctional CD8+ TIL states covering four cancer entities using single-cell chromatin profiling. We map enhancer-promoter interactions in human TILs by integrating single-cell chromatin accessibility with single-cell RNA-seq data from tumor-entity-matching samples and prioritize cell-state-specific genes by super-enhancer analysis. Besides revealing entity-specific chromatin remodeling in exhausted TILs, our analyses identify a common chromatin trajectory to TIL dysfunction and determine key enhancers, transcriptional regulators, and deregulated genes involved in this process. Finally, we validate enhancer regulation at immunotherapeutically relevant loci by targeting non-coding regulatory elements with potent CRISPR activators and repressors. In summary, our study provides a framework for understanding and manipulating cell-state-specific gene-regulatory cues from human tumor-infiltrating lymphocytes.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Humanos , Neoplasias/genética , Sequências Reguladoras de Ácido Nucleico , Regulação da Expressão Gênica , Cromatina/genética , Linfócitos do Interstício Tumoral , Elementos Facilitadores GenéticosRESUMO
Genetic variants that affect mRNA splicing are a major cause of hereditary disorders, but the spliceogenicity of variants is challenging to predict. RNA diagnostics of clinically accessible tissues enable rapid functional characterization of splice-altering variants within their natural genetic context. However, this analysis cannot be offered to all individuals as one in five human disease genes are not expressed in easily accessible cell types. To overcome this problem, we have used CRISPR activation (CRISPRa) based on a dCas9-VPR mRNA-based delivery platform to induce expression of the gene of interest in skin fibroblasts from individuals with suspected monogenic disorders. Using this ex vivo splicing assay, we characterized the splicing patterns associated with germline variants in the myelin protein zero gene (MPZ), which is exclusively expressed in Schwann cells of the peripheral nerves, and the spastin gene (SPAST), which is predominantly expressed in the central nervous system. After overnight incubation, CRISPRa strongly upregulated MPZ and SPAST transcription in skin fibroblasts, which enabled splice variant profiling using reverse transcription polymerase chain reaction, next-generation sequencing, and long-read sequencing. Our investigations show proof of principle of a promising genetic diagnostic tool that involves CRISPRa to activate gene expression in easily accessible cells to study the functional impact of genetic variants. The procedure is easy to perform in a diagnostic laboratory with equipment and reagents all readily available.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Splicing de RNA , Humanos , Splicing de RNA/genética , RNA Mensageiro , Sistema Nervoso Central , EspastinaRESUMO
Dynamic, multi-input gene regulatory networks (GRNs) are ubiquitous in nature. Multilayer CRISPR-based genetic circuits hold great promise for building GRNs akin to those found in naturally occurring biological systems. We develop an approach for creating high-performing activatable promoters that can be assembled into deep, wide, and multi-input CRISPR-activation and -interference (CRISPRa/i) GRNs. By integrating sequence-based design and in vivo screening, we engineer activatable promoters that achieve up to 1,000-fold dynamic range in an Escherichia coli-based cell-free system. These components enable CRISPRa GRNs that are six layers deep and four branches wide. We show the generalizability of the promoter engineering workflow by improving the dynamic range of the light-dependent EL222 optogenetic system from 6-fold to 34-fold. Additionally, high dynamic range promoters enable CRISPRa systems mediated by small molecules and protein-protein interactions. We apply these tools to build input-responsive CRISPRa/i GRNs, including feedback loops, logic gates, multilayer cascades, and dynamic pulse modulators. Our work provides a generalizable approach for the design of high dynamic range activatable promoters and enables classes of gene regulatory functions in cell-free systems.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Regiões Promotoras Genéticas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Redes Reguladoras de Genes , Sistemas CRISPR-Cas/genéticaRESUMO
Human induced pluripotent stem cells (hiPSCs) offer opportunities to study human biology where primary cell types are limited. CRISPR technology allows forward genetic screens using engineered Cas9-expressing cells. Here, we sought to generate a CRISPR activation (CRISPRa) hiPSC line to activate endogenous genes during pluripotency and differentiation. We first targeted catalytically inactive Cas9 fused to VP64, p65 and Rta activators (dCas9-VPR) regulated by the constitutive CAG promoter to the AAVS1 safe harbor site. These CRISPRa hiPSC lines effectively activate target genes in pluripotency, however the dCas9-VPR transgene expression is silenced after differentiation into cardiomyocytes and endothelial cells. To understand this silencing, we systematically tested different safe harbor sites and different promoters. Targeting to safe harbor sites hROSA26 and CLYBL loci also yielded hiPSCs that expressed dCas9-VPR in pluripotency but silenced during differentiation. Muscle-specific regulatory cassettes, derived from cardiac troponin T or muscle creatine kinase promoters, were also silent after differentiation when dCas9-VPR was introduced. In contrast, in cell lines where the dCas9-VPR sequence was replaced with cDNAs encoding fluorescent proteins, expression persisted during differentiation in all loci and with all promoters. Promoter DNA was hypermethylated in CRISPRa-engineered lines, and demethylation with 5-azacytidine enhanced dCas9-VPR gene expression. In summary, the dCas9-VPR cDNA is readily expressed from multiple loci during pluripotency but induces silencing in a locus- and promoter-independent manner during differentiation to mesoderm derivatives. Researchers intending to use this CRISPRa strategy during stem cell differentiation should pilot their system to ensure it remains active in their population of interest.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Miócitos Cardíacos , Células Endoteliais , Diferenciação Celular/genética , EndotélioRESUMO
Haploinsufficiency in retinoic acid induced 1 (RAI1) causes Smith-Magenis syndrome (SMS), a severe neurodevelopmental disorder characterized by neurocognitive deficits and obesity. Currently, curative treatments for SMS do not exist. Here, we take a recombinant adeno-associated virus (rAAV)-clustered regularly interspaced short palindromic repeats activation (CRISPRa) approach to increase expression of the remaining intact Rai1 allele. Building upon our previous work that found the paraventricular nucleus of hypothalamus plays a central role in SMS pathogenesis, we performed paraventricular nucleus of hypothalamus-specific rAAV-CRISPRa therapy by increasing endogenous Rai1 expression in SMS (Rai1±) mice. We found that rAAV-CRISPRa therapy rescues excessive repetitive behavior, delays the onset of obesity, and partially reduces hyperphagia in SMS mice. Our work provides evidence that rAAV-CRISPRa therapy during early adolescence can boost the expression of healthy Rai1 allele and modify disease progression in a mouse model of Smith-Magenis syndrome.
Assuntos
Síndrome de Smith-Magenis , Camundongos , Animais , Síndrome de Smith-Magenis/genética , Síndrome de Smith-Magenis/terapia , Síndrome de Smith-Magenis/metabolismo , Transativadores/genética , Transativadores/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Haploinsuficiência , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Obesidade/genéticaRESUMO
Chromosomal instability (CIN), the process of increased chromosomal alterations, compromises genomic integrity and has profound consequences on human health. Yet, our understanding of the molecular and mechanistic basis of CIN initiation remains limited. We developed a high-throughput, single-cell, image-based pipeline employing deep-learning and spot-counting models to detect CIN by automatically counting chromosomes and micronuclei. To identify CIN-initiating conditions, we used CRISPR activation in human diploid cells to upregulate, at physiologically relevant levels, 14 genes that are functionally important in cancer. We found that upregulation of CCND1, FOXA1 and NEK2 resulted in pronounced changes in chromosome counts, and KIF11 upregulation resulted in micronuclei formation. We identified KIF11-dependent fragilities within the mitotic spindle; increased levels of KIF11 caused centrosome fragmentation, higher microtubule stability, lagging chromosomes or mitotic catastrophe. Our findings demonstrate that even modest changes in the average expression of single genes in a karyotypically stable background are sufficient for initiating CIN by exposing fragilities of the mitotic spindle, which can lead to a genomically diverse cell population.
Assuntos
Instabilidade Cromossômica , Cinesinas , Fuso Acromático , Aneuploidia , Centrossomo/metabolismo , Instabilidade Cromossômica/genética , Aberrações Cromossômicas , Humanos , Cinesinas/genética , Microtúbulos/genética , Mitose/genética , Quinases Relacionadas a NIMA/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismoRESUMO
Healing of large calvarial bone defects remains challenging. An RNA-guided Split dCas12a system is previously harnessed to activate long non-coding RNA H19 (lncRNA H19, referred to as H19 thereafter) in bone marrow-derived mesenchymal stem cells (BMSCs). H19 activation in BMSCs induces chondrogenic differentiation, switches bone healing pathways, and improves calvarial bone repair. Since adipose-derived stem cells (ASCs) can be harvested more easily in large quantity, here it is aimed to use ASCs as an alternative cell source. However, H19 activation alone using the Split dCas12a system in ASCs failed to elicit evident chondrogenesis. Therefore, split dCas12a activators are designed more to co-activate other chondroinductive transcription factors (Sox5, Sox6, and Sox9) to synergistically potentiate differentiation. It is found that co-activation of H19/Sox5/Sox6 in ASCs elicited more potent chondrogenic differentiation than activation of Sox5/Sox6/Sox9 or H19 alone. Co-activating H19/Sox5/Sox6 in ASCs significantly augmented in vitro cartilage formation and in vivo calvarial bone healing. These data altogether implicated the potentials of the Split dCas12a system to trigger multiplexed gene activation in ASCs for differentiation pathway reprogramming and tissue regeneration.
Assuntos
Diferenciação Celular , Condrogênese , RNA Longo não Codificante , Fatores de Transcrição SOXD , Crânio , Fatores de Transcrição SOXD/metabolismo , Fatores de Transcrição SOXD/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Animais , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Tecido Adiposo/citologia , Células-Tronco/metabolismo , Células-Tronco/citologia , Osteogênese/genéticaRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR)-based screening has emerged as a powerful tool for identifying new gene targets for desired cellular phenotypes. The construction of guide RNA (gRNA) pools largely determines library quality and is usually performed using Golden Gate assembly or Gibson assembly. To date, library construction methods have not been systematically compared, and the quality check of each batch has been slow. In this study, an in-house nanopore sequencing workflow was established for assessing the current methods of gRNA pool construction. The bias of pool construction was reduced by employing the polymerase-mediated non-amplifying method. Then, a small gRNA pool was utilized to characterize stronger activation domains, specifically MED2 (a subunit of mediator complex) and HAP4 (a heme activator protein), as well as to identify better gRNA choices for dCas12a-based gene activation in Saccharomyces cerevisiae. Furthermore, based on the better CRISPRa tool identified in this study, a custom gRNA pool, which consisted of 99 gRNAs targeting central metabolic pathways, was designed and employed to screen for gene targets that could improve ethanol utilization in S. cerevisiae. The nanopore sequencing-based workflow demonstrated here should provide a cost-effective approach for assessing the quality of customized gRNA library, leading to faster and more efficient genetic and metabolic engineering in S. cerevisiae.
Assuntos
Sequenciamento por Nanoporos , RNA Guia de Sistemas CRISPR-Cas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ativação Transcricional , Clonagem Molecular , Sistemas CRISPR-Cas/genética , Edição de Genes/métodosRESUMO
BACKGROUND AIMS: Lower back pain is the leading cause of disability worldwide and is often linked to degenerative disc disease (DDD), the breakdown of intervertebral discs. The majority of treatment options for DDD are palliative, with clinicians prescribing medication or physical therapy to return the patient to work. Cell therapies are promising treatment options with the potential to restore functional physiological tissue and treat the underlying causes of DDD. DDD is characterized by biochemical changes in the microenvironment of the disc, including changes in nutrient levels, hypoxia, and changes in pH. Stem cell therapies are promising therapies to treat DDD, but the acidic environment in a degenerating disc significantly hinders the viability of stem cells, affecting their efficacy. Clustered regularly interspaced short palindromic repeats (CRISPR) systems allow us to engineer cell phenotypes in a well-regulated and controlled manner. Recently, CRISPR gene perturbation screens have assessed fitness, growth and provided a means for specific cell phenotype characterization. METHODS: In this study, we use a CRISPR-activation (a) gene perturbation screen to identify gene upregulation targets that enhance adipose-derived stem cell survival in acidic culture conditions. RESULTS: We identified 1213 prospective pro-survival genes and systematically narrowed these down to 20 genes for validation. We further narrowed down our selection to the top five prospective genes using Cell Counting Kit-8 cell viability assays in naïve adipose-derived stem cells and ACAN/Col2 CRISPRa upregulated stem cells. Finally, we examined the extracellular matrix-producing abilities of multiplex ACAN/Col2-pro-survival edited cells in pellet culture. CONCLUSIONS: Using the results from the CRISPRa screen, we are able to engineer desirable cell phenotypes to improve cell viability for the potential treatment of DDD and other disease states that expose cell therapies to acidic environments, while also providing broader knowledge on genes regulating low-pH cell survival.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Humanos , Edição de Genes/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Sobrevivência Celular/genética , Estudos Prospectivos , Concentração de Íons de HidrogênioRESUMO
Hepatocellular carcinoma (HCC) is often diagnosed at an advanced stage and is, therefore, treated with systemic drugs, such as tyrosine-kinase inhibitors (TKIs). These drugs, however, offer only modest survival benefits due to the rapid development of drug resistance. To identify genes implicated in TKI resistance, a cluster of regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 activation screen was performed in hepatoma cells treated with regorafenib, a TKI used as second-line therapy for advanced HCC. The screen results show that Hexokinase 1 (HK1), catalyzing the first step in glucose metabolism, is a top candidate for conferring TKI resistance. Compatible with this, HK1 was upregulated in regorafenib-resistant cells. Using several experimental approaches, both in vitro and in vivo, we show that TKI resistance correlates with HK1 expression. Furthermore, an HK inhibitor resensitized resistant cells to TKI treatment. Together, our data indicate that HK1 may function as a critical factor modulating TKI resistance in hepatoma cells and, therefore, may serve as a biomarker for treatment success.
Assuntos
Carcinoma Hepatocelular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Hexoquinase/metabolismo , Neoplasias Hepáticas/metabolismo , Animais , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Células Cultivadas , Hexoquinase/genética , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos NOD , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Regulação para CimaRESUMO
CRISPR/Cas9-mediated cleavage of DNA, which depends on the endonuclease activity of Cas9, has been widely used for gene editing due to its excellent programmability and specificity. However, the changes to the DNA sequence that are mediated by CRISPR/Cas9 affect the structures and stability of the genome, which may affect the accuracy of results. Mutations in the RuvC and HNH regions of the Cas9 protein lead to the inactivation of Cas9 into dCas9 with no endonuclease activity. Despite the loss of endonuclease activity, dCas9 can still bind the DNA strand using guide RNA. Recently, proteins with active/inhibitory effects have been linked to the end of the dCas9 protein to form fusion proteins with transcriptional active/inhibitory effects, named CRISPRa and CRISPRi, respectively. These CRISPR tools mediate the transcription activity of protein-coding and non-coding genes by regulating the chromosomal modification states of target gene promoters, enhancers, and other functional elements. Here, we highlight the epigenetic mechanisms and applications of the common CRISPR/dCas9 tools, by which we hope to provide a reference for future related gene regulation, gene function, high-throughput target gene screening, and disease treatment.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/genética , Epigênese Genética , DNARESUMO
CRISPR (clustered regularly interspaced short palindromic repeats) activation (CRISPRa) in bacteria is an attractive method for programmable gene activation. Recently, a eukaryote-like, σ54 -dependent CRISPRa system has been reported. It exhibits high dynamic ranges and permits flexible target site selection. Here, an overview of the existing strategies of CRISPRa in bacteria is presented, and the characteristics and design principles of the CRISPRa system are introduced. Possible scenarios for applying the eukaryote-like CRISPRa system is discussed with corresponding suggestions for performance optimization and future functional expansion. The authors envision the new eukaryote-like CRISPRa system enabling novel designs in multiplexed gene regulation and promoting research in the σ54 -dependent gene regulatory networks among a variety of biotechnology relevant or disease-associated bacterial species.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Bactérias/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Eucariotos/genética , Ativação TranscricionalRESUMO
Fibroblast activation is transient in successful wound repair but persistent in fibrotic pathologies. Understanding fibroblast deactivation during successful wound healing may provide new approaches to therapeutically reverse fibroblast activation. To characterize the gene programs that accompany fibroblast activation and reversal during lung fibrosis resolution, we used RNA sequencing analysis of flow sorted Col1α1-GFP-positive and CD45-, CD31-, and CD326-negative cells isolated from the lungs of young mice exposed to bleomycin. We compared fibroblasts isolated from control mice with those isolated at Days 14 and 30 after bleomycin exposure, representing the peak of extracellular matrix deposition and an early stage of fibrosis resolution, respectively. Bleomycin exposure dramatically altered fibroblast gene programs at Day 14. Principal component and differential gene expression analyses demonstrated the predominant reversal of these trends at Day 30. Upstream regulator and pathway analyses of reversing "resolution" genes identified novel candidate antifibrotic genes and pathways. Two genes from these analyses that were decreased in expression at Day 14 and reversed at Day 30, Aldh2 and Nr3c1, were selected for further analysis. Enhancement of endogenous expression of either gene by CRISPR activation in cultured human idiopathic pulmonary fibrosis fibroblasts was sufficient to reduce profibrotic gene expression, fibronectin deposition, and collagen gel compaction, consistent with roles for these genes in fibroblast deactivation. This combination of RNA sequencing analysis of freshly sorted fibroblasts and hypothesis testing in cultured idiopathic pulmonary fibrosis fibroblasts offers a path toward identification of novel regulators of lung fibroblast deactivation, with potential relevance to understanding fibrosis resolution and its failure in human disease.
Assuntos
Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Aldeído-Desidrogenase Mitocondrial/genética , Aldeído-Desidrogenase Mitocondrial/metabolismo , Animais , Bleomicina , Sistemas CRISPR-Cas , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/patologia , Edição de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Pulmão/patologia , Camundongos Transgênicos , RNA-Seq , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Remissão Espontânea , Transdução de Sinais , Fatores de Tempo , TranscriptomaRESUMO
CRISPR-Cas transcriptional programming in bacteria is an emerging tool to regulate gene expression for metabolic pathway engineering. Here we implement CRISPR-Cas transcriptional activation (CRISPRa) in P. putida using a system previously developed in E. coli. We provide a methodology to transfer CRISPRa to a new host by first optimizing expression levels for the CRISPRa system components, and then applying rules for effective CRISPRa based on a systematic characterization of promoter features. Using this optimized system, we regulate biosynthesis in the biopterin and mevalonate pathways. We demonstrate that multiple genes can be activated simultaneously by targeting multiple promoters or by targeting a single promoter in a multi-gene operon. This work will enable new metabolic engineering strategies in P. putida and pave the way for CRISPR-Cas transcriptional programming in other bacterial species.
Assuntos
Engenharia Metabólica , Pseudomonas putida , Sistemas CRISPR-Cas/genética , Escherichia coli/genética , Pseudomonas putida/genética , Ativação Transcricional/genéticaRESUMO
Zika virus (ZIKV) is an arthropod-borne emerging pathogen causing febrile illness. ZIKV is associated Guillain-Barré syndrome and other neurological complications. Infection during pregnancy is associated with pregnancy complications and developmental and neurological abnormalities collectively defined as congenital Zika syndrome. There is still no vaccine or specific treatment for ZIKV infection. To identify host factors that can rescue cells from ZIKV infection, we used a genome-scale CRISPR activation screen. Our highly ranking hits included a short list of interferon-stimulated genes (ISGs) previously reported to have antiviral activity. Validation of the screen results highlighted interferon lambda 2 (IFN-λ2) and interferon alpha-inducible protein 6 (IFI6) as genes providing high levels of protection from ZIKV. Activation of these genes had an effect on an early stage in viral infection. In addition, infected cells expressing single guide RNAs (sgRNAs) for both of these genes displayed lower levels of cell death than did the controls. Furthermore, the identified genes were significantly induced in ZIKV-infected placenta explants. Thus, these results highlight a set of ISGs directly relevant for rescuing cells from ZIKV infection or its associated cell death and substantiate CRISPR activation screens as a tool to identify host factors impeding pathogen infection.IMPORTANCE Zika virus (ZIKV) is an emerging vector-borne pathogen causing a febrile disease. ZIKV infection might also trigger Guillain-Barré syndrome, neuropathy, and myelitis. Vertical transmission of ZIKV can cause fetus demise, stillbirth, or severe congenital abnormalities and neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We used a genome-wide CRISPR activation screen, where genes are activated from their native promoters to identify host cell factors that protect cells from ZIKV infection or associated cell death. The results provide a better understanding of key host factors that protect cells from ZIKV infection and might assist in identifying novel antiviral targets.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Resistência à Doença/genética , Testes Genéticos , Interações Hospedeiro-Patógeno/genética , Infecção por Zika virus/genética , Infecção por Zika virus/virologia , Zika virus/fisiologia , Processamento Alternativo , Expressão Gênica , Testes Genéticos/métodos , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte Proteico , Reprodutibilidade dos Testes , Ativação Viral , Replicação Viral , Infecção por Zika virus/metabolismoRESUMO
BACKGROUND: Fibroblasts regulate tissue homeostasis and the balance between tissue repair and fibrosis. CCAAT/enhancer-binding protein alpha (CEBPA) is a key transcription factor that regulates adipogenesis. CEBPA has been shown to be essential for lung maturation, and deficiency of CEBPA expression leads to abnormal lung architecture. However, its specific role in lung fibroblast regulation and fibrosis has not yet been elucidated. METHODS: Lung fibroblast CEBPA expression, pro-fibrotic and lipofibroblast gene expression were assessed by qRT-PCR. CEBPA gain and loss of function experiments were carried out to evaluate the role of CEBPA in human lung fibroblast activation with and without TGF-ß1 treatment. Adipogenesis assay was used to measure the adiopogenic potential of lung fibroblasts. Finally, CRISPR activation system was used to enhance endogenous CEBPA expression. RESULTS: We found that CEBPA gene expression is significantly decreased in IPF-derived fibroblasts compared to normal lung fibroblasts. CEBPA knockdown in normal human lung fibroblasts enhanced fibroblast pro-fibrotic activation and ECM production. CEBPA over-expression by transient transfection in IPF-derived fibroblasts significantly reduced pro-fibrotic gene expression, ECM deposition and αSMA expression and promoted the formation of lipid droplets measured by Oil Red O staining and increased lipofibroblast gene expression. Inhibition of the histone methyl transferase G9a enhanced CEBPA expression, and the anti-fibrotic effects of G9a inhibition were partially mediated by CEBPA expression. Finally, targeted CRISPR-mediated activation of CEBPA resulted in fibroblasts switching from fibrogenic to lipofibroblast states. CONCLUSIONS: CEBPA expression is reduced in human IPF fibroblasts and its deficiency reduces adipogenic potential and promotes fibrogenic activation. CEBPA expression can be rescued via an inhibitor of epigenetic repression or by targeted CRISPR activation, leading to reduced fibrogenic activation.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Fibroblastos/metabolismo , Edição de Genes , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Adipogenia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibrose , Regulação da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Fenótipo , Interferência de RNA , Transdução de Sinais , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Molecular strategies tailored to promote/correct the expression and/or processing of defective coagulation factors would represent innovative therapeutic approaches beyond standard substitutive therapy. Here, we focus on the molecular mechanisms and determinants underlying innovative approaches acting at DNA, mRNA and protein levels in inherited coagulation factor deficiencies, and in particular on: (i) gene editing approaches, which have permitted intervention at the DNA level through the specific recognition, cleavage, repair/correction or activation of target sequences, even in mutated gene contexts; (ii) the rescue of altered pre-mRNA processing through the engineering of key spliceosome components able to promote correct exon recognition and, in turn, the synthesis and secretion of functional factors, as well as the effects on the splicing of missense changes affecting exonic splicing elements; this section includes antisense oligonucleotide- or siRNA-mediated approaches to down-regulate target genes; (iii) the rescue of protein synthesis/function through the induction of ribosome readthrough targeting nonsense variants or the correction of folding defects caused by amino acid substitutions. Overall, these approaches have shown the ability to rescue the expression and/or function of potentially therapeutic levels of coagulation factors in different disease models, thus supporting further studies in the future aimed at evaluating the clinical translatability of these new strategies.
Assuntos
Transtornos de Proteínas de Coagulação/genética , Transtornos de Proteínas de Coagulação/terapia , Terapia Genética/métodos , Animais , Fatores de Coagulação Sanguínea/genética , Sistemas CRISPR-Cas , DNA/genética , Edição de Genes/métodos , Humanos , RNA Mensageiro/genéticaRESUMO
The rapid advancement of CRISPR technology has enabled targeted epigenome editing and transcriptional modulation in the native chromatin context. However, only a few studies have reported the successful editing of the epigenome in adult animals in contrast to the rapidly growing number of in vivo genome editing over the past few years. In this review, we discuss the challenges facing in vivo epigenome editing and new strategies to overcome the huddles. The biggest challenge has been the difficulty in packaging dCas9 fusion proteins required for manipulation of epigenome into the adeno-associated virus (AAV) delivery vehicle. We review the strategies to address the AAV packaging issue, including small dCas9 orthologues, truncated dCas9 mutants, a split-dCas9 system, and potent truncated effector domains. We discuss the dCas9 conjugation strategies to recruit endogenous chromatin modifiers and remodelers to specific genomic loci, and recently developed methods to recruit multiple copies of the dCas9 fusion protein, or to simultaneous express multiple gRNAs for robust epigenome editing or synergistic transcriptional modulation. The use of Cre-inducible dCas9-expressing mice or a genetic cross between dCas9- and sgRNA-expressing flies has also helped overcome the transgene delivery issue. We provide perspective on how a combination use of these strategies can facilitate in vivo epigenome editing and transcriptional modulation.
Assuntos
Sistemas CRISPR-Cas , Epigênese Genética , Epigenômica , Edição de Genes , Transcrição Gênica , Metilação de DNA , Humanos , Regiões Promotoras GenéticasRESUMO
The inducible CRISPR activation (CRISPR-a) system offers unparalleled precision and versatility for regulating endogenous genes, making it highly sought after in plant research. In this study, we developed a chemically inducible CRISPR-a tool for plants called ER-Tag by combining the LexA-VP16-ER inducible system with the SunTag CRISPR-a system. We systematically compared different induction strategies and achieved high efficiency in target gene activation. We demonstrated that guide RNAs can be multiplexed and pooled for large-scale screening of effective morphogenic genes and gene pairs involved in plant regeneration. Further experiments showed that induced activation of these morphogenic genes can accelerate regeneration and improve regeneration efficiency in both eudicot and monocot plants, including alfalfa, woodland strawberry, and sheepgrass. Our study expands the CRISPR toolset in plants and provides a powerful new strategy for studying gene function when constitutive expression is not feasible or ideal.