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Analyse des voies de régulation de la cardiogenèse et de la différenciation cardiomyocytaire / Analysis of the cardiogenenis pathways and the cardiac differentiationJeziorowska, Dorota 13 December 2016 (has links)
L'objectif général de ce travail de doctorat a été centré sur l'utilisation des cellules pluripotentes induites humaines dans la modélisation et l'évaluation thérapeutique des pathologies cardiaques. Depuis leur découverte en 2006, les iPSC offrent une opportunité pour le développement de modèles cellulaires humains et spécifiques de patients pour l'étude des mécanismes physiopathologiques, l'évaluation de réponses pharmacologiques et le génération de cellules redifférenciées (ici en cardiomyocytes) pour des applications thérapeutiques cellulaires. Dans ce travail nous avons démontré que la quantité mais aussi la qualité finale des cardiomyocytes dérivés d'iPSC dépend des conditions spatiales et pharmacologiques utilisées durant les différentes étapes de différenciation. L'utilisation d'un protocole de différentiation en monocouche avec blocage simultané et transitoire de l'ensemble des voies Wnt (canoniques et non canoniques) permet d'obtenir une maturation plus importante du sarcomère, étape essentielle pour la modélisation des sarcomèropathies La différenciation des iPSC en cardiomyocytes peut aussi être obtenue par une approche moléculaire ciblée visant à activer spécifiquement un programme cardiogénique. Celle-ci est obtenue via l'utilisation d'une protéine Cas9 mutée et couplée à un système transactivateur et permettant le ciblage simultané de 3 facteurs de transcription clés de la cardiogénèse (Gata4, Mef2c et Tbx5). Cette approche moléculaire est renforcée par la combinaison avec une stimulation pharmacologique ciblant la voie Wnt. / The general objective of this work was centered on the use of human induced pluripotent cells in modeling and therapeutic evaluation of cardiac pathologies. Since their discovery in 2006, the iPSC provide an opportunity for the development of human cellular models and specific patients for the study of pathophysiological mechanisms, evaluation of pharmacological responses and the generation redifférenciées cells (cardiomyocytes here) for applications cellular therapeutic. In this work we demonstrated that the quantity but also the final quality of cardiomyocytes derived from iPSC depends on the spatial and pharmacological conditions used during the various stages of differentiation. The use of a monolayer differentiation protocol with simultaneous and transient blocking of all Wnt pathways (canonical and noncanonical) allows to obtain a higher maturation of the sarcomere, an essential step for modeling sarcomeropathies IPSC differentiation into cardiomyocytes can also be obtained by targeted molecular approach to specifically activate cardiogenic program. This is achieved through the use of a mutated Cas9 protein and coupled with transactivator system. This allows simultaneous targeting of 3 key cardiogenesis transcription factors (Gata4, MEF2C and Tbx5). This molecular approach is enhanced by the combination with a pharmacological stimulation targeting the Wnt pathway. Beyond modeling of monogenic cardiac disease, cardiomyocytes derived from iPSC can reproduce more complex and multigenic diseases
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Genome-Wide In Vivo CRISPR Activation Screen to Identify Genetic Drivers of Non-Small Cell Lung Cancer Brain MetastasisAghaei, Nikoo January 2021 (has links)
Brain metastasis (BM), the most common tumor of the central nervous system, occurs in 20-36% of primary cancers. In particular, 20-40% of patients with non-small cell lung cancer (NSCLC) develop brain metastases, with a dismal survival of approximately 4-11 weeks without treatment, and 16 months with treatment. This highlights a large unmet need to develop novel targeted therapies for the treatment of lung-to-brain metastases (LBM). Genomic interrogation of LBM using CRISPR technology can inform preventative therapies targeting genetic vulnerabilities in both primary and metastatic tumors. Loss-of-function studies present limitations in metastasis research, as knocking out genes essential for survival in the primary tumor cells can thwart the metastatic cascade prematurely. However, transcriptional overexpression of genes using CRISPR activation (CRISPRa) has the potential for overcoming dependencies of gene essentiality.
In this thesis, we created and utilized an in vivo genome-wide CRISPRa screening platform to identify novel genes, that when overexpressed, drive LBM. We have developed a patient-derived orthotopic murine xenograft model of LBM using a patient-derived NSCLC cell line (termed CRUK cells) from the Swanton Lab TRACERx study. We introduced a human genome-wide CRISPRa single guide RNA (sgRNA) library into non-metastatic and pro-metastatic lung cancer CRUK cells to achieve 500X representation of each sgRNA in the activation library. We then injected the cells into the lungs of immunocompromised mice and tracked lung tumor development and BM formation. Upon sequencing primary lung tumors and subsequent BM, we will identify enriched sgRNAs which may represent novel drivers of primary lung tumor formation and LBM.
To the best of our knowledge, this study is the first in vivo genome-wide CRISPR activation screen using patient-derived NSCLC cells to help elucidate drivers of LBM. This work serves to provide a framework to gain a deeper understanding of the regulators of BM formation which will hopefully lead to targeted drug discovery that will ultimately be used in clinical trials to help eradicate brain metastasis in NSCLC patients. / Thesis / Master of Science (MSc) / Brain metastasis, or the spread of a primary cancer from another organ to the brain, is the most common adult brain tumor. Brain metastases can arise after the treatment of primary tumors and are only detected in the clinic at a highly malignant stage. Current treatments for brain metastasis consist of surgical removal and palliative chemoradiotherapy, which fail to fully eliminate the brain tumor. Over 20% of cancer patients develop brain metastases, with lung, breast, and skin cancers leading as the top three sources of metastasis. In particular, 40% of patients with non-small cell lung cancer develop brain metastasis, with survival of only 4-11 weeks once diagnosed without treatment, and 16 months with treatment. As systemic therapies for the treatment of non-small cell lung cancer are becoming increasingly effective at controlling primary disease, patients are ironically succumbing to their brain tumors. This highlights a large unmet need to develop novel targeted therapies for the treatment of lung-to-brain metastases (LBM). Functional genomic tools provide the opportunity to investigate the genetic underpinnings of LBM. With the advent of gene editing technologies, we are able to overexpress various genes and observe the impact genetic perturbations have on tumor initiation, growth, and metastasis.
In this thesis, we devised a pre-clinical animal model of LBM that could be used to study genetic drivers of LBM using a gene overexpression tool such that one gene per tumor cell gets activated. We are then able to model the disease trajectory from a lung tumor to brain metastasis development using patient samples in our animal model and identify genes that, upon overexpression, drive LBM. This platform will lead to potential therapeutic targets to prevent the formation of LBM and prolong the survival of patients with non-small cell lung cancer.
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Development of CRISPR-based programmable transcriptional regulators and their applications in plantsSelma García, Sara 01 September 2022 (has links)
[ES] La Biología Sintética de Plantas tiene como objetivo rediseñar las plantas para que adquieran características y funcionalidades novedosas a través de circuitos reguladores ortogonales. Para lograr este objetivo, se deben desarrollar nuevas herramientas moleculares con la capacidad de interactuar con factores endógenos de manera potente y específica. CRISPR/Cas9 surgió como una herramienta prometedora que combina la capacidad personalizable de unión al DNA, a través de la versión catalíticamente inactivada de la proteína Cas9 (dCas9), con la posibilidad de anclar dominios autónomos de activación transcripcional (TADs) a su estructura para lograr una regulación específica de la expresión génica. Los activadores transcripcionales programables (PTAs) pueden actuar como procesadores específicos, ortogonales y versátiles para el desarrollo de nuevos circuitos genéticos en las plantas. En busca de dCas9-PTA optimizados, se llevó a cabo una evaluación combinatoria de diferentes arquitecturas dCas9 con un catálogo de varios TAD. La mejor herramienta resultante de esta comparación, denominada dCasEV2.1, se basa en la estrategia scRNA y la combinación de los dominios de activación EDLL y VPR con un bucle multiplexable gRNA2.1, que es una versión mutada del gRNA2.0 descrito previamente. En este trabajo, el activador programable dCasEV2.1 demostró ser una herramienta potente y específica, logrando tasas de activación más altas que otras estrategias dCas9 disponibles en plantas. Se observaron tasas de activación sin precedentes dirigidas a genes endógenos en N. benthamiana, acompañadas de una estricta especificidad en todo el genoma, lo que hace que esta herramienta sea adecuada para la regulación estricta de redes reguladoras complejas. Como prueba de concepto, se diseñaron cuatro programas de activación para distintas ramas de la ruta de los flavonoides, buscando obtener enriquecimientos metabólicos específicos en hojas de N. benthamiana. El análisis metabólico de las hojas metabólicamente reprogramadas mediante dCasEV2.1 reveló un enriquecimiento selectivo de los metabolitos diana y sus derivados glicosilados, que se correlacionaron con el programa de activación empleado. Estos resultados demuestran que dCasEV2.1 es una herramienta eficaz para la ingeniería metabólica y un componente clave en los circuitos genéticos destinados a reprogramar los flujos metabólicos. Finalmente, basándonos en dCasEV2.1, desarrollamos un sistema optimizado de regulación de genes inducidos por virus (VIGR) que utiliza un vector Potato Virus X (PVX) para el suministro de los programas de activación CRISPR codificados con gRNA. Este enfoque permite controlar el transcriptoma de la planta a través de una aplicación sistémica basada en aerosol de componentes CRISPR a plantas adultas. El nuevo sistema PVX-VIGR produjo una fuerte activación transcripcional en varios genes diana endógenos, incluidos tres factores de transcripción MYB-like seleccionados. Las activaciones específicas de MYB condujeron a perfiles metabólicos distintivos, demostrando que las aplicaciones potenciales de la herramienta dCasEV2.1 en plantas incluyen la obtención de perfiles metabólicos personalizados utilizando un suministro basado en aerosol de instrucciones de reprogramación transcripcional codificadas por gRNA. En resumen, esta tesis proporciona herramientas novedosas para la activación transcripcional fuerte, ortogonal y programable en plantas, con una caja de herramientas ampliada para el suministro de los programas de activación. / [CA] La Biologia Sintètica de Plantes té com objectiu redissenyar les plantes per que obtinguen característiques i funcionalitats innovadores mitjançant circuits reguladors ortogonals. Per arribar a aquest objectiu, s'han de desenvolupar noves ferramentes moleculars amb la capacitat d'interactuar amb factor endògens d'una manera potent i específica. CRISPR/Cas9 va sorgir com una ferramenta prometedora que combina la capacitat personalitzable d'unió al DNA, mitjançant la versió catalíticament inactivada de la proteïna Cas9 (dCas9), amb la possibilitat de fixar dominis autònoms de activació transcripcional (TADs) a la seua estructura per aconseguir una regulació específica de la expressió gènica. Els activadors transcripcionals programables (PTAs) poden actuar com a processadors específics, ortogonals i versàtils per al desenvolupament de nous circuits genètics a les plantes. Buscant dCas9-PTA optimitzats, es va realitzar una avaluació combinatòria de distintes arquitectures dCas9 amb un catàleg de diversos TAD. La millor ferramenta segons aquesta comparació, anomenada dCasEV2.1, es basa en la estratègia scRNA i la combinació del dominis d'activació EDLL i VPR amb un bucle multiplexable gRNA2.1, que es una versió mutada del gRNA2.0 descrit prèviament. En aquest treball, el activador programable dCasEV2.1 es va mostrar com una ferramenta potent i específica, aconseguint nivells d'activació majors que altes estratègies dCas9 disponibles en plantes. Es van observar taxes d'activació sense precedents dirigides a gens endògens en N. benthamiana, junt a una estricta especificitat en tot el genoma, indicant que aquesta ferramenta és adequada per a la regulació estricta de xarxes reguladores complexes. Como proba de concepte, se van dissenyar quatre programes d'activació per a diferent branques de la ruta dels flavonoides, cercant obtenir enriquiments metabòlics específics en fulles de N. benthamiana. L'anàlisi metabòlic de les fulles metabòlicament reprogramades mitjançant dCasEV2.1 va revelar un enriquiment selectiu del metabòlits diana i els seus derivats glicosilats que es correlacionen amb el programa d'activació emprat. Aquests resultats demostren que dCasEV2.1 és una ferramenta eficaç per a l'enginyeria metabòlica i un component clau als circuits genètics destinats a reprogramar els fluxos metabòlics. Finalment, en base a dCasEV2.1, desenvoluparem un sistema optimitzat de regulació de gens induïts per virus (VIGR) que utilitza un vector Potato Virus X (PVX) per al subministrament dels programes d'activació CRISPR codificats amb gRNA. Aquesta aproximació permet controlar el transcriptoma de la planta mitjançant l'aplicació sistèmica basada en aerosol de components CRISPR a plantes adultes. El nou sistema PVX-VIGR va produir una gran activació transcripcional en diversos gens diana endògens, inclosos tres factors de transcripció MYB-like seleccionats prèviament. Les activacions específiques de MYB conduïren a perfils metabòlics distintius, demostrant que les aplicacions potencials de la ferramenta dCasEV2.1 en plantes inclouen la obtenció de perfils metabòlics personalitzats emprant un subministrament basat en aerosol de instruccions de reprogramació transcripcional codificades per gRNA. En resum, aquesta tesis proporciona noves ferramentes per a l'activació transcripcional forta, ortogonal i programable en plantes, amb una caixa de ferramentes eixamplada per al subministraments dels programes d'activació. / [EN] Plant Synthetic Biology aims to redesign plants to acquire novel traits and functionalities through orthogonal regulatory circuits. To achieve this goal, new molecular tools with the capacity of interacting with endogenous factors in a potent and specific manner must be developed. CRISPR/Cas9 emerged as promising tools which combine a customizable DNA-binding activity through the catalytically inactivated version of Cas9 protein (dCas9) with the possibility to anchor autonomous transcriptional activation domains (TADs) to its structure to achieve a specific regulation of the gene expression. The Programmable Transcriptional Activators (PTAs) could act as specific, orthogonal and versatile processor components in the development of new genetic circuits in plants. In search for optimized dCas9-PTAs, a combinatorial evaluation of different dCas9 architectures with a catalogue of various TADs was performed. The best resulting tool of this comparison, named dCasEV2.1, is based on the scRNA strategy and the combination of EDLL and VPR activation domains with a multiplexable gRNA2.1 loop, which is a mutated version of the previously described gRNA2.0. In this work, the dCasEV2.1 programable activator was proved to be a strong and specific tool, achieving higher activation rates than other available dCas9 strategies in plants. Unprecedented activation rates were observed targeting endogenous genes in N. benthamiana, accompanied by strict genome-wide specificity that makes this tool suitable to perform a tight regulation of complex regulatory networks. As a proof of concept, a design of four activation programs to activate different branches of the flavonoid pathway and obtain specific metabolic enrichments in N. benthamiana leaves was performed. The metabolic analysis on the dCasEV2.1 metabolically reprogrammed leaves revealed a selective enrichment of the targeted metabolites and their glycosylated derivatives that correlated with the activation program employed. These results demonstrate that dCasEV2.1 is a powerful tool for metabolic engineering and a key component in genetic circuits aimed at reprogramming metabolic fluxes. Finally, based on dCasEV2.1, we developed an optimized Viral Induced Gene Regulation (VIGR) system that makes use of a Potato Virus X (PVX) vector for the delivery of the gRNA-encoded CRISPR activation programs. This approach offers a way to control the plant transcriptome through a spray-based systemic delivery of CRISPR components to adult plants. The new PVX-VIGR system led to strong transcriptional activation in several endogenous target genes, including three selected MYB-like transcription factors. Specific MYB activations lead to distinctive metabolic profiles, showing that the potential applications of the dCasEV2.1 tool in plants include the obtention of custom metabolic profiles using a spray-based delivery of gRNA-encoded transcriptional reprogramming instructions. In sum, this thesis provides novel tools for strong, orthogonal and programmable transcriptional activation in plants, with an expanded toolbox for the delivery of the activation programs. / Selma García, S. (2022). Development of CRISPR-based programmable transcriptional regulators and their applications in plants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/185046 / TESIS
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Identification of novel active Cas9 orthologs from metagenomic dataDemozzi, Michele 12 April 2022 (has links)
CRISPR-Cas is the state-of-the-art biological tool that allows precise and fast manipulation of the genetic information of cellular genomes. The translation of the CRISPR-Cas technology from in vitro studies into clinical applications highlighted a variety of limitations: the currently available systems are limited by their off-target activity, the availability of a Cas-specific PAM sequence next to the target and the size of the Cas protein. In particular, despite high levels of activity, the size of the CRISPR-SpCas9 editing machinery is not compatible with an all-in-one AAV delivery system and the genomic sequences that can be targeted are limited by the 3-NGG PAM-dependency of the SpCas9 protein. To further expand the CRISPR tools repertoire we turned to metagenomic data of the human microbiome to search for uncharacterized CRISPR-Cas9 systems and we identified a set of novel small Cas9 orthologs derived from the analysis of reconstructed bacterial metagenomes. In this thesis study, ten candidates were chosen according to their size (less than 1100aa). The PAM preference of all the ten orthologs was established exploiting a bacterial-based and an in vitro platform. We demonstrated that three of them are active nucleases in human cells and two out of the three showed robust editing levels at endogenous loci, outperforming SpCas9 at particular targets. We expect these new variants to be very useful in expanding the available genome editing tools both in vitro and in vivo. Knock-out-based Cas9 applications are very efficient but many times a precise control of the repair outcome through HDR-mediated gene targeting is required. To address this issue, we also developed an MS2-based reporter platform to measure the frequency of HDR events and evaluate novel HDR-modulating factors. The platform was validated and could allow the screening of libraries of proteins to assess their influence on the HDR pathway.
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The CRISPR-Cas systemStens, Cassandra, Enoksson, Isabella, Berggren, Sara January 2020 (has links)
Derived from and inspired by the adaptive immune system of bacteria, CRISPR has gone from basic biology knowledge to a revolutionizing biotechnological tool, applicable in many research areas such as medicine, industry and agriculture. The full mechanism of CRISPR-Cas9 was first published in 2012 and various CRISPR-Cas systems have already passed the first stages of clinical trials as new gene therapies. The immense research has resulted in continuously growing knowledge of CRISPR systems and the technique seems to have the potential to greatly impact all life on our planet. Therefore, this literature study aims to thoroughly describe the CRISPR-Cas system, and further suggest an undergraduate laboratory exercise involving gene editing with the CRISPR-Cas9 tool. In this paper, we describe the fundamental technical background of the CRISPR-Cas system, especially emphasizing the most studied CRISPR-Cas9 system, its development and applications areas, as well as highlighting its current limitations and ethical concerns. The history of genetic engineering and the discovery of the CRISPR system is also described, along with a comparison with other established gene editing techniques. This study concludes that a deeper knowledge about CRISPR is important and required since the technique is applicable in many research areas. A laboratory exercise will not only inspire but also provide extended theoretical and practical knowledge for undergraduate students.
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