Whole genome doubling (WGD) generates genetically unstable tetraploid cells that fuel tumorigenesis. Cells that undergo WGD must acquire adaptive characteristics to accommodate their tetraploid state, and these adaptations may confer unique vulnerabilities that can be exploited therapeutically. We analyzed the genomes of ~9,700 primary human cancer samples to uncover genetic alterations that are specifically enriched in WGD+ cancer cells. Through integrating our genetic analysis with gene essentiality data acquired from Project Achilles, we identified gene dependencies in WGD+ cells. Moreover, we identified genes that are essential for the viability of WGD+ cancer cells, but non-essential to non-transformed diploid cells. We demonstrated that the gene encoding for the mitotic kinesin KIF18A is dispensable for mitosis in diploid cells, but becomes critical for accurate chromosome segregation and viability in WGD+ cells, making it an attractive drug target. Collectively, this work revealed new strategies to specifically target WGD+ cancer cells, namely targeting the gene KIF18A, while sparing the normal diploid cells from which they arise. / 2022-06-04T00:00:00Z

Markéta Sypecká CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis Introduction: Chronic lymphocytic leukemia (chronic lymphoid leukemia, CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent lymphocytes. CLL is the most common form of leukemia found in adults in Western countries. Course of the disease can differ: some patients die rapidly, within 2-3 years of diagnosis, because of complications from CLL, but most patients live 5-10 years. However, every stage of this disease has significantly higher level of miR-155, which is known as oncomiR. Micro RNAs represent negative regulators of gene expression. MiR-155 affects genes, which are involved in leukemogenesis and cell cycle. And it is known, that miR-155 suppresses its targets. We hypothesized that by gene editing of CLL B - cells we unblock miR-155 targets and find out correlation between these targets (known and unknown) with CLL leukemogenesis. Method we use for gene editing is CRISPR/Cas9, which enables to delete sequence of mature miR-155 in genome of leukemic B-cells. Methods: CRISPR/Cas9, nucleofection, qRT-PCR, FACS Results:We achieved to isolate clone that bears one allelic deletion (miR-155-/+) in sequence for mature...

Les inflammasomes sont des complexes protéiques impliqués dans l’immunité innée, qui sont activés par de multiples signaux de danger. Des mutations héréditaires des protéines de l’inflammasome peuvent être responsables de son activation excessive et in fine de la survenue de pathologies auto-inflammatoires chez l’être humain. À l’heure actuelle, aucun modèle cellulaire ne permet d’étudier spécifiquement la dynamique d’activation des inflammasomes et de préciser les conséquences des mutations activatrices sur celles-ci. J’ai donc généré un modèle humain macrophagique exprimant une protéine recombinante FLAG3x-ASC endogène, commune aux différents inflammasomes dans la lignée cellulaire humaine monocytaire/macrophagique THP-1. Cette lignée a été générée par édition génique par la technologie CRISPR-Cas9 en utilisant un substrat de recombinaison permettant d’insérer la séquence codant pour le FLAG3X in frame du locus PYCARD codant pour ASC. J’ai pu générer 6 clones FLAG3x-ASC dans la lignée THP-1. Les clones générés ont été validés en confirmant l’expression et la fonctionnalité de la protéine recombinante FLAG3x-ASC et en vérifiant l’absence de mutations indésirables hors-cible générée par la nucléase Cas9. Une fois ce modèle généré, j’ai pu également reproduire un variant génétique du gène NLRC4, protéine sensor de l’inflammasome du même nom, retrouvé chez un patient présentant une maladie auto-inflammatoire. La validation des clones mutant pour NLRC4 est en cours. Ce projet permettra la caractérisation de la dynamique d’activation de l’inflammasome dans un modèle physiologique et pathologique. Ceci permettra une avancée importante dans la compréhension de l’inflammasome et son agrégation ainsi que la régulation de ce complexe face aux signaux de danger. / Inflammasomes are multiproteic complexes that are involved in innate immunity and are activated by multiple signals of dangers. Hereditary mutations in inflammasome components lead to its excessive activation that is responsible for human auto-inflammatory disease. While these mutations are supposed to alter the dynamic of inflammasome activation, there is no current human model allowing the dynamic study of this complex. I generated a human cellular model expressing an endogenous FLAG3x ASC protein, an adaptator protein common to several inflammasomes, in the human monocytic/macrophagic THP-1 cell line. This model was created through CRISPR-Cas9 genome engineering using a recombination template allowing the in frame integration of the sequence encoding the FLAG3X peptide at the PYCARD locus encoding ASC. I generated and validated the expression and the functionality of 6 FLAG3x-ASC THP-1 cell lines. Furthermore, these cell lines are devoided of CRISPR-Cas9 off-target effect. In this model, I further reproduced a genetic variant of the inflammasome component NLRC4 observed in a patient presenting with autoinflammatory manifestation. The functional validation of the FLAG3x-ASC THP-1 harboring the NLRC4 variant is on-going. This project will allow to study the dynamic of the activation of the inflammasome in healthy and pathological conditions. Those results will help refine our comprehension of inflammasome complexation and regulation in response to danger signals.

No description available.

Neisseria meningitidis, a commensal β-proteobacterium residing exclusively in the human nasopharynx, is a leading cause of sepsis and epidemic meningitis worldwide. While comparative genome analysis was able to define hyperinvasive lineages that are responsible for most of the cases of invasive meningococcal disease (IMD), the genetic basis of their virulence remains unclear. Recent studies demonstrate that the type II C CRISPR/Cas system of meningococci is associated with carriage and less invasive lineages. CRISPR/Cas, an adaptive defence system against foreign DNA, was shown to be involved in gene regulation in Francisella novicida. This study shows that knockout strains of N. meningitidis lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells in a strain-dependant manner, which constitutes a central step in the pathogenesis of IMD. Consequently, this study indicates that the meningococcal CRISPR/Cas system fulfils functions beyond the defence of foreign DNA and is involved in the regulation of meningococcal virulence. / Neisseria meningitidis, ein ß-Proteobakterium, welches als Kommensale ausschließlich den humanen Nasopharynx besiedelt, ist ein weltweit führender Verursacher von Sepsis und epidemischer Meningitis. Auch wenn mittels vergleichender Genomanalysen hyperinvasive Stämme definiert werden konnten, welche für die meisten Fälle von invasiven Meningokokkenerkrankungen verantwortlich sind, bleibt die genetische Grundlage ihrer Virulenz ungeklärt. In vorangegangenen Studien konnte gezeigt werden, dass das Typ II-C CRISPR/Cas-System der Meningokokken assoziiert ist mit Trägerstämmen. CRISPR/Cas ist ein adaptives Verteidigungssystem der Bakterien gegen fremde DNA, das darüber hinaus Aufgaben in der Genregulation von Francisella novicida erfüllt. Diese Arbeit zeigt, dass knockout Stämme von N. meningitidis, denen das Cas9-Protein fehlt, in Abhängigkeit von ihrem genetischen Hintergrund die Fähigkeit verlieren an Zellen des menschlichen Nasopharynx zu adhärieren. Die Adhäsion an den Wirtszellen stellt einen zentralen Schritt in der Pathogenese der invasiven Meningokokkenerkrankungen dar. Die Ergebnisse dieser Arbeit deuten darauf hin, dass das CRISPR/Cas-System in Meningokokken neben seiner Funktion als bakterielles Immunsystem an der Regulation der bakteriellen Virulenz beteiligt sein könnte.

No description available.

La technologie d’édition CRISPR peut être définie comme un outil biologique qui par son efficacité, son extrême précision et la facilité de sa modélisation, permet aujourd’hui de modifier le génome des organismes vivants en général et celui de l’homme en particulier. Sa découverte en 2012 par les chercheuses française et américaine, E. Charpentier et J. Doudna, récompensées par le Prix Nobel de chimie 2020, permet des applications thérapeutiques, au niveau germinal, pour des maladies à transmission autosomique dominante. Jusqu’ici, aucune technologie antérieure d’édition génomique, ni aucun diagnostic anténatal (DPI, DPN), n’avait été capable de prévenir ces maladies. En novembre 2018, le chercheur chinois Hé Jiankui annonce avoir utilisé la technologie CRISPR pour éditer des embryons humains viables. Selon Jiankui cette tentative consiste à modifier génétiquement des embryons humains en FIV afin de prévenir « définitivement » l’infection au VIH des futurs bébés. Cette modification génétique est ainsi transmissible à leur descendance. A partir de là, il s’est ouvert un tournant décisif de l’édition du génome humain héritable. Celui-ci s’apparente, dans notre contexte marqué par la convergence des NBIC, à une perspective transhumaniste. Car à la vérité, CRISPR n’aurait pas fait que prévenir l’infection au VIH chez ces bébés, il aurait surtout amélioré un caractère génétique conférant à ces derniers une immunité à vie contre le VIH-SIDA, avec pour principal corollaire, que de telles modifications sont héritables. Cette application non thérapeutique controversée, nous a conduit à nous demander successivement s’il est souhaitable de se servir de la technologie d’édition du génome CRISPR-Cas9, pour corriger au niveau germinal ou embryonnaire, une anomalie génétique afin de préserver le futur enfant de certains handicaps qui pourraient mettre en péril sa santé ou alourdir sa vie ?Jusqu’où de telles modifications pourraient être jugées comme bénéfiques ou à risque pour l’enfant et qui en aurait l’ultime légitimité d’en juger ?Peut-on alors affirmer, qu’en regard de l’extrême étroitesse qui existe entre la finalité thérapeutique d’une modification génomique germinale ou embryonnaire et l’amélioration/l’augmentation génétique, le risque d’altération de la nature humaine et de fait, la sortie hors de l’espèce humaine devient inéluctable ? / Doctorat en Philosophie / info:eu-repo/semantics/nonPublished

<div> <div> <div> <p>Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene- editing as they do not require sequence motifs adjacent to their targets for function. One promising pAgo candidate from the halophilic archaeon Natronobacterium gregoryi (NgAgo) has been the subject of intense debate regarding its potential in eukaryotic systems. NgAgo was initially claimed to edit genes in mammalian cells, but the report was retracted due to replication failure. Due to low solubility, subsequent studies refolded NgAgo and suggested that it cuts RNA but not DNA; however, mutation of the conserved active site does not abolish cleavage activity, raising the possibility of nuclease contamination. Another independent study demonstrated gene-editing via NgAgo in bacteria. These inconsistent results underscore the knowledge gap and roadblock for NgAgo-based gene-editing tool development. <br><br> </p><div> <div> <div> <p>In this work, I revisit this enzyme and characterize its function in vitro and in a bacterial system. The halophilic features of NgAgo have been neglected in the literature, leading to inconclusive results. Like other halophilic proteins, NgAgo has modified amino acid composition, leading to failure of domain identification/function prediction via sequence alignment. Indeed, using more sensitive structural alignments, I identified a new single-stranded DNA binding domain, repA, in NgAgo and other halophilic pAgos. Due to its halophilic nature, NgAgo expresses poorly in low-salt environments, with the majority of protein being insoluble and inactive even after refolding. However, soluble NgAgo indeed cuts DNA. NgAgo DNA-cleaving activity can only be abolished via mutation in the canonical PIWI domain and repA deletion, revealing a new catalytic behavior in pAgos. Moreover, NgAgo requires both repA and PIWI domains to create double- stranded DNA breaks, leading to cell death or enhancing homologous recombination, or gene- editing, at a modest level in bacteria. Rational protein engineering of NgAgo was also pursued to increase solubility. Although three out of seven mutants showed significant increases in solubility, they lost the ability to cleave DNA in E.coli. Structural modeling revealed some subtle but important differences in the protein structures, explaining why the mutants lose their function. Besides, a selection system for improving endonuclease activity was optimized for future pAgo optimization. Collectively, this work revealed that NgAgo possesses unique catalytic behavior in the pAgo family and has some gene-editing application potential. More importantly, this work expands knowledge of the pAgo family, providing a foundation for future pAgo-based gene- editing tool development. </p> </div> </div> </div> </div> </div> </div>

Die Rolle von Connexinen und Gap Junction-vermittelter Kommunikation in pluripotenten Stammzellen sowie der frühen Embryonalentwicklung sind bis heute nicht vollständig aufgeklärt. Mutationen in humanen Connexinen verursachen eine Vielzahl von Krankheiten. Connexin-defiziente iPS Zellen stellen eine gute Basis für die Erforschung der Rolle von Connexinen während der Embryonalentwicklung und bei der Krankheitsentstehung dar. Das Ziel der vorliegenden Arbeit war es, das CRISPR/Cas9-System in pluripotenten Stammzellen erfolgreich anzuwenden und ein Protokoll zur Erstellung verschiedener Cx43-Defektmutanten zu entwerfen. Nach der Etablierung der CRSIPR/Cas9-Methode in HEK293T-Zellen konnte in der vorliegenden Arbeit darüber hinaus erfolgreich eine Cx43-Defizienz in FSiPS-Zellen erzeugt werden. Weiterhin wurden mehrere Cx43-Mutanten geschaffen und initial auf Pluripotenzmarker und ihr Differenzierungspotential untersucht. Diese Arbeit bildet die Basis für weitere Untersuchungen des Cx43 in iPS-Zellklonen und davon abgeleiteten Zelltypen sowie artifiziellen 3D-Gewebekulturen. Darüber hinaus bildet sie die Grundlage für die Bildung weiterer Connexin-Defektmutanten sowie von iPS-Zellen mit krankheitsrelevanten Mutationen. / The roles of connexins and gap junction-mediated communication in pluripotent stem cells and early embryonic development have not been fully elucidated to date. Mutations in human connexins cause a variety of diseases. Connexin-deficient iPS cells provide a good basis for studying the role of connexins during embryonic development and in disease development. The aim of the present work was to successfully apply the CRISPR/Cas9 system in pluripotent stem cells and to design a protocol to generate different Cx43 defective mutants. Furthermore, after establishing the CRSIPR/Cas9 method in HEK293T cells, a Cx43 deficiency in FSiPS cells was successfully generated. Furthermore, several Cx43 mutants were created and initially screened for pluripotency markers and their differentiation potential. This work forms the basis for further studies of Cx43 in iPS cell clones and derived cell types as well as artificial 3D tissue cultures. Furthermore, it forms the basis for the generation of further connexin defect mutants as well as iPS cells with disease-relevant mutations.

The discovery of antibiotics have revolutionized modern medicine, facilitating the treatment of a variety of bacterial infections, enabled surgeries otherwise impossible to perform and increased life expectancy in all countries. However, the rapid development of resistance among microorganisms and the increasing numbers of clinical outbreaks caused by multiresistant bacteria have accelerated the need for new alternatives to antibiotics. Probiotic bacteria armed with defense systems have been studied as potential substitutes of antibiotics. These probiotic competitors can still contribute to the spread of resistance genes among microorganisms through horizontal gene transfer. The aim of this study was to investigate whether constructed CRISPR-Cas9 systems have the potential to protect probiotic bacteria against horizontal transfer of antibiotic resistance genes. Transformation, transduction and conjugation assays in strains carrying or not carrying a plasmid-bourne CRISPR-Cas9 system were performed in order to compare the frequencies of transfer of the most common resistance genes. The transformation and transduction assays demonstrated that the constructed CRISPR-Cas9 system entails a decrease in efficiency of transfer for targeted resistance genes. Moreover, it can be concluded that potentially increasing Cas9 levels by reducing its degradation results in increased prevention of horizontal gene transfer through transformation and transduction. Finally, we state that the CRISPRCas9 system does not result in protection against antibiotic resistance genes entering the cells through conjugation.