Spelling suggestions: "subject:"CRISPR/cas9."" "subject:"CRISPR/has9.""
161 |
CRISPR-Cas9 Mediated Restoration of Dystrophin Expression and Inhibition of Myostatin: A Novel Gene Therapy for Duchenne Muscular DystrophyRangan, Apoorva 01 January 2016 (has links)
Duchenne Muscular Dystrophy (DMD) is an X-linked recessive genetic disease, caused by a frame-shift mutation in the dystrophin gene. Current gene therapies for DMD target dystrophin transcripts in existing skeletal and cardiac muscle, rather than adipose and fibrotic tissues. These approaches may be unable to repair muscle functionality in DMD patients who have already undergone extensive muscle damage and wasting. Thus, successful DMD therapies must consider the underlying genetic cause and pathology. Inhibition of the gene myostatin, a negative regulator of muscle growth, has been shown to ameliorate muscle loss. Here, the CRISPR-Cas9 gene-editing platform is proposed to restore dystrophin expression and inhibit myostatin as a novel gene therapy in DMD patient derived induced pluripotent stem cells. Successful CRISPR-Cas9 mediated gene editing would be determined using PCR amplification, western blot analysis, immunofluorescence staining, and off target sequence analysis in differentiated skeletal muscle cells.
|
162 |
Caratterizzazione del gene LIPOSSIGENASI 4 e approccio CRISPR-Cas9 per aumentare la resistenza alla fusariosi di mais / LIPOXYGENASE 4 CHARACTERIZATION AND CRISPR-CAS9 APPROACH TO ENHANCE FUSARIUM VERTICILLIOIDES (FV) RESISTANCE IN ZEA MAYSBORRELLI, VIRGINIA MARIA GRAZIA 14 December 2018 (has links)
Il Fusarium verticillioides (Fv) causa il marciume rosa della spiga e contamina le cariossidi con fumonisine, una famiglia di micotossine che colpisce mangimi e alimenti considerata cancerogena per l'uomo e gli animali. Sono stati condotti diversi studi per identificare i geni del mais associati alla resistenza della pianta ospite all'infezione da Fv e l'accumulo di fumonisina. È noto che le ossilipine regolano la difesa contro i patogeni e che il cross-talk lipidico ospite-patogeno influenza la patogenesi. A questo proposito, i mutanti di mais trasposonici del gene ZmLOX4, la linea suscettibile W22 e la resistente TZI18 sono stati testati per la resistenza a Fv mediante il saggio biologico Rolled Towel Assay (RTA). Inoltre, sono stati studiati i profili di espressione di 16 geni coinvolti nella via LOX e volatili verdi (GLV) e l'attività della lipossigenasi è stata analizzata nelle stesse linee. Inoltre, è stata applicata la tecnologia di modifica del genoma di Clustered Shortspeed Palindromic Repeat / Cas9 associato (CRISPR / Cas9) regolarmente esaminata per indagare le possibili implicazioni del gene ZmLOX6 e del fattore di trascrizione ZmWRKY125 nei meccanismi di resistenza contro Fv. L'espressione di questi geni è stata precedentemente osservata dagli esperimenti di RNA - Seq in genotipi resistenti al mais e Studi di Genome Wide Association (GWAS) che hanno portato a un SNP significativamente associato a ZmWRKY125. Inoltre, il gene ZmLOX4 è stato overespresso nella linea A188 per valutare un possibile miglioramento della resistenza alla malattia verso Fv. Il lavoro molecolare del CRISPR si basa su una doppia clonazione utilizzando due diverse single guide RNA (sgRNA) per un bersaglio genico. I costrutti sotto il promotore ZmpUBI nel vettore binario p1609 sono stati trasformati nella linea A188 utilizzando la trasformazione mediata da Agrobacterium tumefaciens. Le piante di mais modificate nei geni ZmLOX6 e ZmWRKY125 e ZmLOX4 che sovraesprimono saranno caratterizzate per RTA, prove sperimentali in campo e per il loro contenuto di fumonisina. Inoltre, saranno testati l’attività lipossigenasica totale, i suoi metaboliti derivati e le osslipine, oltre all'analisi dell'espressione dei principali geni coinvolti nella via dell'acido jasmonico. / Fusarium verticillioides (Fv) causes ear rot in maize and contaminates the kernels with fumonisins, a family of mycotoxins that affects feed and food and considered carcinogenic for humans and animals. Several studies were conducted to identify maize genes associated with host plant resistance to Fv infection and fumonisin accumulation. It is known that plant lipoxygenase (LOX)-derived oxylipins regulate defense against pathogens and that the host-pathogen lipid cross-talk influences the pathogenesis. In this regard, maize mutants carrying Mu insertions in the ZmLOX4 gene, the susceptible W22 and the resistant TZI18 lines were tested for Fv resistance by the screening method rolled towel assay (RTA). Additionally, the expression profiles of 16 genes involved in the LOX and green leaves volatiles (GLV) pathway were studied and the lipoxygenase activity was investigated in the same lines as well.
Furthermore, the genome editing technology of Clustered Regularly Interspaced Short Palindromic Repeat/associated Cas9 (CRISPR/Cas9) was applied in order to investigate the possible implication of the lipoxygenase gene ZmLOX6 and the transcription factor ZmWRKY125 in the resistance mechanisms against Fv. The enhanced expression of these genes was previously observed by RNA - Seq experiments in maize resistant genotypes and Genome Wide Association Studies (GWAS) resulted in one SNP significantly associated with ZmWRKY125. Moreover, the gene ZmLOX4 was over-expressed in the line A188 for evaluating a possible improvement of the disease resistance towards Fv. The CRISPR cloning was based on a double cloning using two different guides (sgRNA) for one gene target. The constructs under the maize promoter ZmpUBI in the binary vector p1609 were transformed into the maize A188 line using Agrobacterium tumefaciens mediated transformation. Maize plants edited in the genes ZmLOX6 and ZmWRKY125, and over-expressing ZmLOX4 will be characterized for Fv resistance using rolled towel assay, field assay and for their fumonisin content. Furthermore, the content of jasmonic acid, its derivative metabolites, and oxylipins will be tested, as well as the expression analysis of the main genes involved in the jasmonic acid pathway will be performed.
|
163 |
Molecular regulation and function of Gata2 in the programming of haemogenic endotheliumDobrzycki, Tomasz January 2017 (has links)
Haematopoietic stem cells (HSCs) maintain the vertebrate blood system throughout life. Exploiting their clinical potential requires a thorough understanding of the natural origins of the HSCs. They first arise from the haemogenic endothelium (HE), located in the main embryonic artery, the dorsal aorta. Our understanding of the genetic mechanisms underlying HE specification remains incomplete, but one of the crucial transcription factors is Gata2. We found that a conserved enhancer of zebrafish gata2a gene (i4 enhancer) is active in vivo specifically in endothelial cells, including the HE. To unravel the function of gata2a in specifying the HSCs, we have targeted the i4 enhancer with CRISPR/Cas9, generating the first reported genomic deletion of an endogenous cis-regulatory region in zebrafish. Deletion of the i4 enhancer leads to a decrease in endothelial gata2a expression and a concomitant transient decrease in the number of HSCs. This is marked by an early decrease in the expression of gata2b, a gata2a paralogue previously shown to be required for the initiation of the haematopoietic programme. Our results suggest non-redundant roles of both zebrafish gata2 paralogues in programming of HSCs, providing insights into different roles of GATA2 throughout the programming of HSCs. We also confirmed the previously reported loss of HSCs upon MO-mediated knockdown of lmo4a, associated with increased gata2a expression in HE. We validated the increase in gata2a levels in TALEN-generated lmo4a mutants. To identify the links between lmo4a, gata2a and the HE programming, we have profiled the transcriptome of lmo4a-deficient endothelial cells, including the HE. Our results suggest that Lmo4a may be a global regulator of the transcriptional programming of the HE. Moreover, Wnt signalling pathway may regulate gata2a downstream of lmo4a. This provides novel insights into the gene regulatory network orchestrating the generation of HSCs in the embryo.
|
164 |
Genome-scale identification of cellular pathways required for cell surface recognitionSharma, Sumana January 2018 (has links)
A range of biochemically diverse molecules located in the plasma membrane— such as proteins, glycans, and lipids—mediate cellular recognition events, initiation of signalling pathways, and the regulation of processes important for the normal development and function of multicellular organisms. Interactions mediated by cell surface receptors can be challenging to detect in biochemical assays, because they are often highly transient, and membrane-embedded receptors are difficult to solubilise in their native conformation. The biochemical features of low-affinity extracellular protein interactions have therefore necessitated the development of bespoke methods to detect them. Here, I develop a genome-scale cell-based genetic screening approach using CRISPR-Cas9 knockout technology that reveals cellular pathways required for specific cell surface recognition events. Using a panel of high-affinity monoclonal antibodies, I first establish a method from which I identify not only the direct receptor but also other required gene products, such as co-receptors, post-translational modi cations, and transcription factors contributing to antigen expression and subsequent antibody-antigen recognition on the surface of cells. I next adapt this method to identify cellular factors required for receptor interactions for a panel of recombinant proteins corresponding to the ectodomains of cell surface proteins to the endogenous surface receptors present on a range of cell lines. In addition to finding general cellular features recognised by many ectodomains, I also identify direct interaction partners of recombinant protein probes on cell surfaces together with intracellular genes required for such associations. Using this method, I identify IGF2R as a binding partner for the R2 subunit of GABAB receptors, providing a mechanism for the internalisation and regulation of GABAB receptor signalling. The results here demonstrate that this single approach can identify the molecular nature and cell biology of surface receptors without the need to make any prior assumptions regarding their biochemical properties.
|
165 |
CRISPR-Cas9 mediated HMGCL KO in 3xTg AD mice reduces the cognitive deficit improvement seen in an intermittent metabolic switching regimenKil, Eric Joon Bum 01 January 2018 (has links)
Individuals in modern Western societies are experiencing increasing sedentary lifestyles, overindulgence of high fat, high-sugar diets, and extremely sterilized conditions, putting immense pressure on researchers and clinicians alike to come up with viable treatments for conditions implicated with an aging society. Emerging research have published the benefits of IMS and metabolic switching in a variety of neuroprotective, cellular stress resistance, and neuroplasticity pathways in animal models and clinical results from randomized trials of IMS regimens with susceptible human populations are soon to be published. The application of genome editing and next-generation sequencing (NGS) strategies to clinical and neurodegenerative research continues to elucidate the relationship between a patient’s specific genetic background and modern environmental stressors towards disease pathology. This study attempts to utilize novel CRISPR/Cas9 strategies to introduce targeted gene edits and explores the role of reduced ketone-body synthesis/metabolism with 3-hydroxymethyl-3-methylglutaryl-CoA lyase HMGCL KO, in the therapeutic and neuroprotective potential of intermittent metabolic switching in 3xTg mice, genetically predisposed for Alzheimer pathology. IMS-mediated attenuation of hippocampal spatial memory deficits was confirmed in 5-month-old 3xTg mice using Morris Water Maze and Aβ1-40, Aβ1-42, total tau and p-tau levels were calculated accordingly. Mice receiving time-restricted feeding (TRF) and caloric restriction (CR) regardless of KO performed better in the hippocampal-dependent spatial memory test and ELISA analysis of CSF revealed reduced p-tau levels of 3xTg WT TRF + CR mice relative to WT control or the two experimental groups. Overall, genetic modifications of key metabolic enzymes highlight the variable therapeutic results of the glucose to ketone metabolic switch on cognitive deficits depending on an organism’s genetic background.
|
166 |
Axonal target specificity in the CRISPR/Cas9 era : a new role for Reelin in vertebrate visual sytem development / Spécificité du ciblage axonale dans l'ère du CRISPR/Cas9 : un rôle nouveau pour la Reelin pendant le développement du système visuel chez les vertébrésDi Donato, Vincenzo 16 September 2016 (has links)
Les connexions neuronales du système visuel forment des synapses spatialement distribuées en couches discrètes. Comprendre la base du ciblage spécifique axonale est critique pour déchiffrer la formation des réseaux neuronaux complexes. Dans une première étude, nous avons investigué le rôle de la protéine de la matrice extracellulaire Reelin dans la formation in vivo du circuit rétinotectal chez le poisson zèbre. Ce circuit se compose de cellules ganglionnaires de la rétine (CGRs) transmettant l’information visuelle au cerveau via la projection de leur axone dans les différentes couches du tectum optique. Nous avons démontré que la Reelin secrétée par de neurones inhibiteurs localisés dans les couches supérieures du tectum optique forme un gradient. L’induction de mutations délétères dans la voie de signalisation canonicale de la Reelin à l’aide d’outils génétiques a conduit à des défauts de ciblage des axones de CGRs. Nos résultats démontrent un nouveau rôle de la Reelin lors du développement du système visuel et la décrivent comme signature moléculaire nécessaire au ciblage et au positionnement précis des axones de CGRs.Dans une seconde étude, nous avons utilisé la technique CRISPR/Cas9 pour développer une nouvelle approche de mutagénèse conditionnelle chez le poisson zèbre. Nos résultats démontrent que la perturbation de gènes dans des tissues spécifiques peut être effectué par l’induction de l’expression de la protéine Cas9 via le système Gal4/UAS. Nous avons établis un outil pour induire l’apparition de mutations délétères dans des clones de cellules mais aussi dans des cellules individuelles, tous pouvant être suivit distinctement grâce à un marquage génétique. / Neuronal connections in the visual system are arranged in synaptic laminae. Understanding the basis of lamina-specific axonal targeting is critical to gain deeper insights on how complex neural networks form. In a first study we investigated the role of the ECM protein Reelin during zebrafish retinotectal circuit formation in vivo. Here retinal ganglion cells (RGCs) convey the visual information to the brain by projecting their axons to different layers of the optic tectum. We demonstrated that Reelin secreted by a specific class of tectal superficial inhibitory neurons is spatially distributed in a superficial-to-deep gradient within the tectal neuropil. Induced gene disruption for all the components of the canonical Reelin pathway expressed in the retinotectal system resulted in aberrant layering of RGC axons suggesting a role for Reelin pathway in axonal sublaminar segregation. Altogether our findings elucidate a new role for Reelin in vertebrate visual system development, during which it acts as molecular cue by imparting positional information for ingrowing RGCs.In a second study we took advantage of the CRISPR/Cas9 technology to develop a novel approach for conditional mutagenesis in zebrafish. Our results provide evidence that tissue-specific gene disruption can be achieved by driving Cas9 expression with the Gal4/UAS system. We established a tool to induce loss-of-function mutations in cell clones or single cells that can be followed by genetic labeling, enabling their phenotypic analysis. Our technique has the potential to be applied to a wide-range of model organisms, allowing systematic mutagenesis and labeling on a genome-wide scale.
|
167 |
Functional characterization of the biological significance of the ZBED6/ZC3H11A locus in placental mammalsYounis, Shady January 2017 (has links)
The recent advances in molecular and computational biology have made possible the study of complicated transcriptional regulatory networks that control a wide range of biological processes and phenotypic traits. In this thesis, several approaches were combined including next generation sequencing, gene expression profiling, chromatin and RNA immunoprecipitation, bioinformatics and genome editing methods in order to characterize the biological significance of the ZBED6 and ZC3H11A genes. A mutation in the binding site of ZBED6, located in an intron of IGF2, disrupts the binding and leads to 3-fold upregulation of IGF2 mRNA in pig muscle tissues. The first part of the thesis presents a detailed functional characterization of ZBED6. Transient silencing of ZBED6 expression in mouse myoblasts led to increased Igf2 expression (~2-fold). ChIP-seq analysis of ZBED6 and histone modifications showed that ZBED6 preferentially binds active promoters and modulates their transcriptional activities (paper I). In the follow-up studies using CRISPR/Cas9 we showed that either the deletion of ZBED6 or its binding site in Igf2 (Igf2ΔGGCT) led to more than 30-fold up-regulation of Igf2 expression in myoblasts. Differentiation of these genetically engineered cells resulted in hypertrophic myotubes. Transcriptome analysis revealed ~30% overlap between the differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with significant enrichment of muscle-specific genes. ZBED6-overexpression in myoblasts led to cell cycle arrest, reduced cell viability, reduced mitochondrial activities and impaired the differentiation of myoblasts (paper II). Further studies on cancer cells showed that ZBED6 influences the growth of colorectal cancer cells with dramatic changes in the transcription of hundreds of cancer-related genes (paper III). The phenotypic characterization of Zbed6-/- and Igf2pA/mG mouse models showed that the ZBED6-Igf2 axis has a major effect on regulating muscle growth and the growth of internal organs. Transcriptome analysis demonstrated a massive up-regulation of Igf2 expression (~30-fold) in adult tissues, but not in fetal tissues, of transgenic mice (paper IV). In the second part of the thesis we investigated the cellular function of Zc3h11a, the gene harboring ZBED6 in one of its first introns. The function of the ZC3H11A protein is so far poorly characterized. We show that ZC3H11A is a novel stress-induced protein that is required for efficient mRNA export from the nucleus. The inactivation of ZC3H11A inhibited the growth of multiple viruses including HIV, influenza, HSV and adenoviruses (paper V).
|
168 |
Funkční charakterizace proteinů rodiny Alba u huseníčku rolního / Functional characterization of Alba-family genes in Arabidopsis thalianaKočová, Helena January 2020 (has links)
(anglicky) Alba-family proteins were identified in Archaea and Eucarya and are classified among the oldest and the most conserved nucleic acid-binding proteins. The binding preferences and roles differ among certain evolution clades. In Crenarchaea they represent chromatin-binding proteins, while their role in RNA metabolism is suggested in Euryarchaea and Eukaryotes. ALBA proteins are well characterized in human, where they play a role in the RNAse P/MRP complex and in unicellular parasites, such as Plasmodium and Trypanosoma, where an involvement in the life cycle regulation is confirmed. In plants, their role is not yet well understood. The aim of this thesis is to increase a knowledge about the Alba-family proteins in the model plant Arabidopsis thaliana. Based on a minimal changes to development and reproduction in single mutants and high sequence similarity, a functional redundancy of the proteins was assumed. For better understanding of the ALBA proteins function, three smaller members of the family were edited by the same metod. The obtained triple mutant showed delay in flowering. ALBA dimer formation was confirmed in many organisms. BiFC method was used to determine Arabidopsis ALBA homodimerization. The data analysis showed potential homodimerization in most of them.
|
169 |
Dynamika de novo DNA metylace a její vliv na expresi transgenu a CRISPR/Cas9 mutagenezi / Dynamics of de novo DNA methylation and its impact on transgene expression and CRISPR/Cas9 mutagenesisPřibylová, Adéla January 2021 (has links)
Genetic information must be protected, maintained and copied from cell to daughter cells, from generation to generation. In plants, most of the cells contain complete genetic information, and many of these cells can regenerate to a whole new plant. Such a feature leads to the need for precise control of which genes will be active and which not because in growth and differentiation, only the activity of specific genes for the individual cells, tissues, organs are required. One of the mechanisms controlling the gene activity is RNA interference (RNAi), which down- regulates or blocks the expression of specific genes at the transcriptional or post-transcriptional level. The crucial part of the RNAi is guiding the RNAi machinery to the target. It is mediated via sequence complementarity of the target with a small RNA (sRNA), which is diced from a double- stranded RNA (dsRNA) precursor. The molecular mechanism of dsRNA and sRNA formation and also the target origin predestinates the subsequent silencing pathway. In transcriptional gene silencing (TGS), the gene expression is regulated through chromatin epigenetic modifications. One of the epigenetic marks is cytosine methylation, which is established mainly by RNA-directed DNA-methylation (RdDM) pathway. Although the protein machinery was relatively...
|
170 |
Depletion of recombination-specific cofactors by the C-terminal mutant of the activation-induced cytidine deaminase causes the dominant negative effect on class switch recombination / AIDのC末端変異体は特異的共役因子を枯渇させるため、クラススイッチ組換えにドミナントネガティブ効果を及ぼすAl, Ismail Azza Darwish 26 March 2018 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21030号 / 医科博第91号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 生田 宏一, 教授 清水 章, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
|
Page generated in 0.0606 seconds