Dissertations / Theses on the topic 'Système CRISPR-Cas9'

Le cancer est responsable de plus de 15% des décès. L’activation d’oncogènes et l’inactivation de gènes suppresseurs de tumeur contribuent à la transformation des cellules. Dans 50% des cas de cancers le gène TP53 est muté. C’est pourquoi comprendre les conséquences de ces mutations est indispensable pour développer des tests permettant de cibler p53 dans le cadre de thérapies. Dans cette étude nous avons utilisé la nouvelle technique de modification de génomes, CRISPR-Cas9. Cette technique a été utilisée dans le but d’introduire des mutations spécifiques de TP53 dans le génome de fibroblastes non tumoraux. Nous avons alors analysé les effets de ces mutations au niveau transcriptionnel et protéomique. Ces analyses aideront à identifier les effets spécifiques de chaque mutation de p53. Ces résultats seront utilisés pour établir des lignées cellulaires permettant de cribler et d’identifier des composés capables de restaurer la fonction sauvage de p53
Cancer is responsible for more than 15% of human deaths. Activation of oncogenes and inactivation of tumor suppressor genes contribute to malignant transformation of cells. Mutations of the tumor suppressor gene TP53 are observed in about 50% of human cancers. Therefore, it is of high interest to understand functional consequences of TP53 mutations in order to develop biological tests that allow targeting mutant p53 for oncotherapy. In this study we use CRISPR-Cas9, the latest genome editing technique, for introducing specific TP53 mutations into the genome of a non-tumoral fibroblast cell line. We analyze the effects of p53 mutations at the transcriptomic and proteomic level. These analyses will help identifying gene- and pathway-specific effects of distinct p53 mutations. These results will be used for establishing cell lines that allow high throughput screening, in order to discover new chemical compounds that are able to restore crucial functions of mutant p53 proteins

Le système CRISPR-Cas9 a révolutionné le monde de la génétique. Il est aujourd’hui utilisé dans de nombreux domaines de recherches comme la médecine, l’agronomie, l’environnement etc. Il commence également à être utilisé en clinique. Cependant, depuis quelques années, de plus en plus d’études soulignent les risques génotoxiques de la nucléase Cas9. Alors que les premières études s’intéressaient au manque de spécificité et aux risques hors cibles du système et que des solutions ont été apportées, les nouvelles constatations pointent du doigt les risques de génotoxicité au locus ciblé. En effet, des risques d’apparition d’insertions/délétions non voulue au locus lors d’expériences d’HDR, d’inversion de séquences, de larges délétions chromosomiques terminales ont été décrits. Le travail de thèse présenté ici a pour but de trouver des solutions contre ces évènements génotoxiques au locus ciblé. Dans un premier temps, nous avons développé des solutions dans les lignées cellulaires, et les cellules souches hématopoïétiques en développant le système nickase, puis nous nous sommes intéressés aux cellules humaines pluripotentes induites avec l’utilisation d’un guide allèle-spécifique. Enfin, nous avons mis au point un système sensible de détection des risques génotoxiques dans des cellules diploïdes immortalisées afin de mieux les caractériser. Bien que très efficace, CRISPR-Cas9 nucléase reste un outil à optimiser. Des contrôles qualités doivent être mis en place pour une utilisation correcte de ce nouvel outil révolutionnaire pour la biologie et ses limites doivent être connues pour mieux les maitriser
CRISPR-Cas9 system has revolutionized genetic world. Nowadays, it is used in various research domains as medicine, agronomy, environment… It is also involved in clinic. However, for a few years, more and more studies have underlined the Cas9 genotoxicity risks. As the first studies focused on the system lack of specificity and on its off-target risks, solutions were brought. Now, new ascertainments emphasize the on-target genotoxic risks. Indeed, non-desired insertions / deletions at the locus in HDR experiments, sequence inversions, large chromosomic truncations were described. The thesis work presented here, aims at finding solutions against these on-target genotoxic risks. In a first time, we have developed solutions in cell lines and hematopoietic stem cells with the nickase system development, and then we have focused on human induced pluripotent stem cells with the use of an allele-specific guide. Finally, we have worked out in sensitive detection genotoxic risks system in immortalized diploid cells to characterized them better. Quality controls must be set up to a correct use of this new biologic revolutionary tool and its limits must be known to controlled them better

Leishmania est un parasite eucaryote divergent responsable d’un large spectre de maladies à travers le monde. Ce parasite est caractérisé par une aneuploïdie mosaïque, constitutive, c’est-à-dire qu’au sein d’une population chaque cellule comporte une combinaison unique de mono-, di- et trisomies de chacun de ses 36 chromosomes. L’aneuploïdie mosaïque est générée et maintenue chez les générations suivantes grâce à un taux élevé de répartition asymétrique des chromosomes lors de la mitose, entrainant le gain ou la perte de chromosomes entiers. Ceci implique une régulation non-conventionnelle de la réplication, suivie d’une ségrégation permissive des chromosomes.L’objectif général de cette étude était de comprendre la dynamique de la réplication de l’ADN ainsi que de cartographier les sites d’initiation de la réplication chez Leishmania, en utilisant la technique du peignage moléculaire d’une part et celle du ChIP-seq d’une autre part. Nous avons ainsi pu caractériser les différents paramètres de progression de la fourche de réplication. Un des résultats majeurs qui ressort de cette étude est que Leishmania possède les plus grandes distances inter-origines et la plus grande vitesse de réplication parmi les autres eucaryotes déjà étudiés. Nous avons également pu estimer que le génome de Leishmania possède environ 168 origines de réplication. Afin d’étudier les acteurs impliqués dans la réplication de l’ADN chez Leishmania, nous avons développé l’outil génétique CRISPR/Cas9. Pour développer cet outil, nous avons basé notre approche sur une stratégie à deux vecteurs : l’un permet l’expression du single guide (sg)RNA et l’autre celle de l’endonucléase Cas9. La validation de cet outil génétique a été réalisée par le knock-out du locus PFR2 codant une protéine flagellaire. Dans un second temps, nous avons fait évoluer le CRISPR/Cas9 vers un système inductible pour réaliser les knock-out et des étiquetages au locus endogène de protéines d’intérêt. Nous avons utilisé ce nouveau système pour étudier la fonction de deux protéines potentiellement impliquées dans le complexe de reconnaissance des origines de réplication. Malgré une fuite du système, nous avons pu réaliser le KO des gènes Orc1b et Orc1/Cdc6 et suivre la progression du cycle cellulaire. Nous avons pu constater que la perte de ces gènes conduisait à un défaut de croissance ainsi qu’à l’apparition de cellules sans noyau. L’insertion d’une étiquette au locus endogène d’Orc1b nous a parmi de confirmer la localisation que nous avions obtenue avec une construction épisomale et va permettre d’étudier plus précisément le rôle de cette protéine.En conclusion, nous avons mis en évidence des paramètres de réplication originaux et démontré, en utilisant le CRISPR/Cas9, que les protéines Orc1b et Ocr1/Cdc6 étaient impliquées dans la duplication du noyau de Leishmania, ce qui est en accord avec leur rôle putatif dans la réplication de l’ADN
Leishmania, a protozoan parasite which causes a large range of diseases worldwide, is characterized by a constitutive 'mosaic aneuploidy', i.e. each cell in a population possesses a unique combination of mono-, di- and trisomies for each of its 36 heterologous chromosomes. Mosaic aneuploidy is generated and maintained via high rates of asymmetric chromosomal allotments during mitosis, leading to the gain or loss of whole chromosomes. This implies an unconventional regulation of the replication, followed by a permissive segregation.The main objective of this study was to unravel DNA replication dynamics and to map the replication initiation sites in Leishmania using DNA combing and ChIP-seq analyses. First, we have characterized DNA replication fork parameters. One of the major findings of this study was that Leishmania exhibits the fastest replication speed and the largest interorigin distances among the eukaryotes tested so far. We have also estimated that the Leishmania major genome possesses 168 origins of replication.To study the actors involved in DNA replication, we first had to develop novel genetic tools. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated endonuclease 9) system is a recently discovered powerful technique for genome editing. In order to adapt this system to Leishmania, we have chosen a two-plasmid strategy: one for the expression of the single guide (sg) RNA and a second for the expression of the endonuclease CAS9. The proof of concept has been based on the disruption of the paraflagellar rod-2 (PFR2) loci by the CRISPR-Cas9 system. In a second attempt, we have developed an inducible CRISPR-Cas9 system, both to obtain knock outs and to perform marker-free endogenous gene tagging. We used the system to investigate the function of Origin Recognition Complex proteins. Although the system was leaky, the genome was edited as expected. We thus deleted Orc1b and Orc1/Cdc6 and monitored the cell cycle progression of the parasite. We found that the depletion of these nuclear proteins lead to a growth defect and to the appearance of zoids (anucleated cells). The endogenous tagging of Orc1b confirmed the localization previously obtained using an episomal expression vector, and will allow further investigation on the role of this protein.In total, we have shown the presence of original replication dynamics parameters in Leishmania, and using CRISPR Cas9, we have demonstrated that Orc1b and Orc1/Cdc6 are involved in the nuclear duplication of Leishmania, in agreement with their putative in DNA replication

Les bactériophages sont des maîtres manipulateurs, prenant le contrôle complet d'une cellule bactérienne, contournant les systèmes de défense et détournant la machinerie de transcription et de traduction du génome bactérien pour la réplication virale. Les grandes étapes de la multiplication des phages sont connues, pourtant les mécanismes intrinsèques de cette prise de contrôle restent un mystère bien préservé. Malgré la petite taille et la simplicité relative des génomes de phages, seuls les gènes associés à la structure des virions sont amplement caractérisés. Toutefois, les protéines non structurales qui sont susceptibles d'être responsables de la prise de contrôle sont rarement étudiées. C'est effectivement le cas pour le phage modèle 2972, infectant la souche Streptococcus thermophilus DGCC7710 largement utilisée en industrie laitière. Son génome code pour 44 protéines putatives, dont 14 protéines sont classées comme étant non structurales et dont aucune fonction n'est encore associée. Lors de ce projet de maîtrise, le système CRISPR-Cas de type II-A, naturellement présent chez S. thermophilus, a été utilisé à des fins d'édition du génome de ce phage pour étudier le rôle des protéines non structurales. Ce système est idéal pour les manipulations génétiques des phages virulents généralement complexes à modifier. Ainsi, une connaissance approfondie des interactions entre le phage et son hôte sera un outil indispensable pour développer de meilleures méthodes de contrôle des phages en industrie laitière.
Bacterial viruses are master manipulators of bacterial cells. They are able to take complete control of a bacterium, bypassing bacterial immune systems, hijacking core transcription and translation machinery, and typically resulting in lysis of the host. Although the major steps of phage replication are well understood, very little is known about the mechanisms of the host-cell takeover. Despite phages having relatively small and 'simple' genomes, generally only the structural proteins have been well characterized. In contrast, non-structural proteins, which include those involved in host cell takeover, tend to be completely uncharacterized. This is certainly the case for the model of Streptococcus thermophilus phages, 2972, which infects the strain DGCC7710 widely used by the dairy industry. Its genome encodes for 44 putative proteins, 14 of which are non-structural and have no known function. In this master thesis, the type II-A CRISPR-Cas system naturally present in S. thermophilus was used for genome engineering purposes to investigate the role of non-structural proteins of phage 2972. This natural bacterial immune system provides an ideal means for genetic manipulation of virulent phages, which are otherwise intractable. This could lead to potentially valuable discoveries allowing us to further fine-tune the bacteria used in various biological processes.

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

Le fonctionnement du cerveau repose sur le transfert d’informations de neurone en neurone au niveau de la synapse. Ce transfert d’informations n’est pas immuable et une synapse peut être potentialisée ou inhibée. Des travaux réalisés ces 20 dernières années ont notamment identifié les astrocytes, un autre type de cellules présentes dans le cerveau, comme des partenaires clefs des neurones capables de réguler la transmission synaptique. Plusieurs voies de signalisation, comme la recapture des ions, des neurotransmetteurs et la sécrétion de facteurs solubles, permettent aux astrocytes d’influencer le fonctionnement des synapses. Cependant, il existe une autre voie, encore peu explorée, qui pourrait participer à la régulation de la transmission synaptique par les astrocytes : la libération de vésicules extracellulaires.Les vésicules extracellulaires (VE) sont de petites particules délimitées par une membrane et qui contiennent des molécules bioactives telles que des protéines, des acides nucléiques et des lipides. La plupart des types cellulaires produisent ces vésicules, qui leur permettent d’échanger des composants avec d’autres cellules plus ou moins distantes. Les VE sont impliquées dans de nombreux processus, comme le fonctionnement du système immunitaire ou la progression de maladies neurodégénératives. Bien que certains travaux rapportent que les astrocytes sont capables de libérer des VE, nous ne savons toujours pas si ces vésicules d’origine astrocytaire sont importantes pour les fonctions synaptiques.L’objectif de cette thèse est d’identifier si les vésicules extracellulaires libérées par les astrocytes jouent un rôle clef dans la régulation de la transmission et de la plasticité synaptique. Pour répondre à cette problématique, nous avons imaginé deux approches complémentaires.Dans un premier temps, nous avons isolé les VE sécrétées par des astrocytes in vitro afin d’en étudier les conditions de libération et le contenu. Nous avons montré d’une part qu’une stimulation avec de l’ATP induit une augmentation de la quantité de petites VE libérées par les astrocytes sur une période de 30 minutes. De plus le contenu en microARN des VE libérées est modifié par cette même stimulation à l’ATP. Une analyse bioinformatique des cibles de ces microARNs nous a permis de prédire que ce changement de contenu pourrait affecter, entre autres, les voies de signalisation régulant la transmission synaptique dans les cellules réceptrices. Ces modifications semblent être spécifiquement induites par l’ATP, car une exposition au glutamate, un neurotransmetteur excitateur, ou au GABA, un neurotransmetteur inhibiteur, ne modifient pas la quantité de petites VE libérées par les astrocytes en l’espace de 30 minutes.Dans un deuxième temps, nous avons étudié l’implication des VE d’origine astrocytaire dans la transmission synaptique in vivo. Pour cela, nous avons créé un outil permettant d’inhiber la sécrétion de petites VE par les astrocytes, chez la souris adulte. En effet, l’enzyme Cas9 est connue pour sa capacité à agir comme des ciseaux moléculaires, coupant l’ADN à des endroits soigneusement choisis afin de modifier le génome. Nous avons tiré profit de cette capacité et dirigé la Cas9 contre un gène impliqué dans la biogénèse des petites VE, afin d’invalider ce gène et donc d’inhiber la libération de ces vésicules. Ce système est mis en œuvre spécifiquement dans les astrocytes adultes grâce à une approche virale conçue sur mesure. Grâce à cet outil, nos résultats préliminaires montrent que l’inhibition de la libération de petites VE d’origine astrocytaire altère la plasticité synaptique dans l’hippocampe de souris mâles.En conclusion, nos résultats suggèrent que les petites vésicules extracellulaires libérées par les astrocytes pourraient effectivement être impliquées dans la régulation de certaines formes de plasticité synaptique, et qu’il serait intéressant d’explorer d’avantage cette voie de recherche
Brain function relies on the transfer of information between neurons, which occurs at a subcellular structure called the synapse. Interestingly, the efficiency of a synapse can be modified under certain conditions, potentiating or inhibiting information transfer. Over the past 20 years, astrocytes, a type of glial cells, have been identified as key neuronal partners that are able to regulate synaptic transmission. Several pathways allowing astrocytes to regulate synaptic function have already been elucidated, such as for instance ion clearance, neurotransmitter recycling or release of soluble factors. Another interesting but under-investigated pathway would be through the release of extracellular vesicles.Extracellular vesicles (EVs) are small membrane-bound particles that contain bioactive molecules such as proteins, nucleic acids, and lipids. Most cells release these vesicles which allow them to exchange cellular components with neighbouring -but also sometimes distant- cells. EVs have been linked to many biological processes, such as immune function or spreading of neurodegenerative diseases. Some studies suggest that astrocytes also release EVs, yet it is still unclear whether these astrocyte-derived vesicles are involved in synaptic functions.This thesis aims at elucidating whether astrocyte-derived extracellular vesicles play a key role in the regulation of synaptic transmission and plasticity. To address this question, we have designed two complementary studies.We first isolated astrocyte-derived EVs in vitro in order to investigate their release rate and their content. We have shown on one hand that exposing astrocyte cultures to ATP in vitro leads to an increase in the amount of small EVs released within 30 min of the stimulus. Furthermore, the microRNA content of these vesicles is altered in response to the stimulus. A bioinformatics analysis predicted that the altered EV content could eventually affect signalling pathways involved in synaptic transmission in recipient cells. These changes seem to be induced specifically by ATP, since exposure to the excitatory neurotransmitter glutamate or to the inhibitory neurotransmitter GABA did not modify the amount of small EVs released within 30 min of the stimulation.We also studied the involvement of astrocyte-derived EVs in vivo. To this end, we developed a tool that allows us to inhibit EV release from astrocytes in the adult mouse brain. Our tool uses the Cas9 enzyme, i.e. the well-known molecular scissors that can specifically cut DNA at a chosen locus in order to modify an organism’s genome. We used Cas9 to invalidate a gene involved in EV biogenesis, thereby inhibiting small EV release. We specifically implemented the Cas9 system in astrocytes using a custom-designed viral approach. Using this tool, our preliminary data suggest that inhibiting the release of small EVs from astrocytes alters a form synaptic plasticity in the hippocampus of adult male mice.To conclude, our findings suggest that small astrocyte-derived extracellular vesicles could indeed be involved in the regulation of some forms of synaptic plasticity and will hopefully encourage further studies to understand the underlying mechanisms

L’étude de la dynamique de conjugaison des plasmides conjugatifs chez les bactéries Gram-négatif est la thématique centrale de recherche de notre laboratoire et autour de laquelle s’est articulé mon projet de thèse. Mes travaux de recherche ont eu pour but d’apporter de véritables connaissances sur l’étendue et l’impact de la conjugaison dans les communautés bactériennes. Le biofilm est largement considéré par la communauté scientifique comme un hotspot favorisant le transfert de gènes principalement en raison des contacts cellulaires propices qui existent dans sa structure. Or, les seules études qui ont essayé de démontrer expérimentalement que le biofilm permet d’augmenter les transferts par conjugaison n’apportent aucunes données claires sur la dynamique de ces transferts qui ont lieu dans le biofilm et comment celui-ci impacte ces transferts. L’approche que nous avons utilisée pour étudier la dynamique de conjugaison dans le biofilm repose sur un projet collaboratif entre notre laboratoire et celui du Dr Knut Drescher, basé au Biozentrum de Bâle en Suisse. Cette collaboration a permis de déployer des techniques de microscopie à fluorescence innovantes développées par nos deux laboratoires et jusqu’ici jamais utilisées dans le contexte de l’étude de la conjugaison dans le biofilm.Nous nous sommes centrés sur le plasmide RP4 qui est un plasmide conjugatif de type IncP. Retrouvé au sein de nombreux environnements naturels, il a été le modèle plasmidique principal des études qui se sont intéressées à la conjugaison dans le biofilm, et il a été largement exploité comme outil génétique par la communauté scientifique. Malgré le fait qu’il ait été très utilisé, les mécanismes de transferts du plasmide RP4 sont très peu décrits. Le plasmide RP4 s’est donc révélé comme un modèle d’étude de la conjugaison très pertinent que nous avons utilisé à la fois dans un aspect biotechnologique pour élargir le spectre d’hôte des systèmes antibactériens TAPs et à la fois dans un aspect fondamental pour étudier sa dynamique de conjugaison, que ce soit au sein d’une population E. coli cultivées en 2D et au sein d’une population E. coli structurées en biofilm 3D.Durant mes travaux de thèse, j’ai donc exploité le plasmide RP4 pour véhiculer des systèmes CRISPR antibactériens chez diverses espèces bactériennes phylogénétiquement éloignées. J’ai apporté les premières images en temps réel du transfert du plasmide RP4 en 2D et de nouvelles données très intéressantes sur la chronologie de conversion de l’ADN en double brin dans la receveuse. Enfin, une approche totalement innovante a permis d’étudier la dynamique de conjugaison du plasmide RP4 dans le biofilm. Ces résultats constituent finalement la première étude qui décrit réellement comment la conjugaison a lieu dans le biofilm et qui va au-delà en termes de compréhension sur cette dynamique grâce à l’approche 2D que nous avions mis en place. Nous démontrons que biofilm n’est pas un hotspot pour le transfert du plasmide RP4 et que les facteurs de la matrice EPS qui compose sa structure n’empêchent pas la dissémination du plasmide. Mais qu’il s’agit plutôt du stade de développement du biofilm qui va rendre possible l’accessibilité des donneurs à l’attachement aux zones de contact avec la surface, à proximité des cellules receveuses
The study of conjugation dynamics of conjugative plasmids in Gram-negative bacteria is the central research theme of our laboratory and around which my thesis project was built. The aim of my research was to provide real knowledge on the extent and impact of conjugation in bacterial communities. The biofilm is widely considered by the scientific community as a hotspot for gene transfer mainly because of the favorable cell contacts that exist in its structure. However, the only studies that have attempted to demonstrate experimentally that biofilms increase gene transfer by conjugation do not provide clear data on the dynamics of these transfers that take place in the biofilm and how the biofilm impacts these transfers. The approach we used to study the dynamics of conjugation in biofilm is based on a collaborative project between our laboratory and that of Dr. Knut Drescher, based at the Biozentrum in Basel, Switzerland. This collaboration allowed us to deploy innovative fluorescence microscopy techniques developed by our two laboratories and never used before in the context of the study of conjugation in biofilm.We focused on the RP4 plasmid which is an IncP conjugative plasmid. Found within many natural environments, it has been the primary plasmid model for studies that have focused on conjugation in the biofilm, and has been widely exploited as a genetic tool by the scientific community. Despite the fact that it has been widely used, the transfer mechanisms of the RP4 plasmid are very poorly described. The RP4 plasmid has thus proven to be a very relevant model for studying conjugation that we have used both in a biotechnological aspect to broaden the host spectrum of antibacterial TAPs systems and in a fundamental aspect to study its conjugation dynamics, both within a 2D cultured E. coli population and within a 3D biofilm structured E. coli population.During my thesis work, I therefore exploited the RP4 plasmid to carry antibacterial CRISPR systems in various phylogenetically distant bacterial species. I provided the first real-time images of the RP4 plasmid transfer in 2D and very interesting new data on the timing of DNA double-strand conversion in the recipient. Finally, a totally innovative approach allowed to study the conjugation dynamics of the RP4 plasmid in the biofilm. These results finally constitute the first study that really describes how conjugation takes place in the biofilm and that goes beyond in terms of understanding this dynamic thanks to the 2D approach that we had set up. We demonstrate that biofilm is not a hotspot for the transfer of the RP4 plasmid and that the factors of the EPS matrix that compose its structure do not prevent the dissemination of the plasmid. Rather, it is the stage of biofilm development that makes it possible for the donors to attach to the surface contact areas near the recipient cells

La maladie d’Alzheimer est la plus commune des formes de démence qui touche presque cinquante millions de personnes dans le monde. Les symptômes les plus fréquents sont la perte de mémoire, la difficulté à planifier des tâches et des confusions temporelles et spatiales. Il n’existe à ce jour aucun traitement pour cette maladie. La protéine précurseur de l’amyloïde (APP) est habituellement coupée par l’enzyme alpha-sécrétase, cependant une coupure anormale par la bêta-sécrétase conduit à la formation de peptides bêta-amyloïdes, qui forment des agrégats s’accumulant sous forme de plaques dans le cerveau des patients Alzheimer. De nombreuses mutations du gène APP sont à l’origine de changements dans la séquence d’acides aminés de la protéine, responsable d’une accumulation accrue de plaques. Ces mutations sont appelées formes familiales de la maladie d’Alzheimer ou FAD (Familial Alzheimer’s disease). Cependant il a été découvert qu’une forme de FAD d’APP (mutation islandaise A673T) présente dans une population d’Europe nordique, différant d’une seule paire de bases du gène normal dans l’exon 16, modifiant une alanine de la protéine en thréonine qui réduit de 40% sa coupure par la bêta-sécrétase. La découverte de la technologie CRISPR/Cas9 ouvre de nouvelles perspectives pour le développement de traitements préventifs ou curatifs des maladies génétiques et dans notre cas Alzheimer. L’endonucléase Cas9 peut couper l’ADN double brin du génome en étant guidée par un ARNg et en reconnaissant une séquence cible « protospacer » suivie d’un PAM (protospacer adjacent motif). Depuis 2016, une optimisation du système CRISPR appelée édition de base permet maintenant de modifier de façon très précise la base cytidine enthymine et les guanines en adénines sur le brin opposé de l’ADN. Le premier article de cette thèse vise à démontrer que l’ajout de la forme FAD A673Ten codominance avec une autre forme pathologique provoque ou non des répercussions bénéfiques sur la sécrétion de peptides amyloïde-beta. Les expériences ont été réalisées avec des plasmides, permettant de visualiser un effet maximum de la mutation A673T. Il était important de déterminer si cette mutation était protectrice en codominance pour assurer une approche thérapeutique la plus polyvalente possible. Nous avons confirmé cet effet bénéfique sur une majorité de formes FAD et en particulier sur la mutation London V717I.Le deuxième article de cette thèse traite de l’introduction de la mutation A673T par un système dérivé de CRISPR/Cas9, l’édition de base. Plusieurs modèles d’éditeurs de base ont été comparés et optimisés dans le but d’obtenir une modification du génome aussi efficace et précise que possible. Un candidat a été sélectionné après des tests sur cellules modèles HEK 293T et neuroblastomes SH-SY5Y.La troisième partie de ce manuscrit présente les résultats obtenus lors de l’insertion de cet éditeur de base dans des vecteurs viraux dans le but d’infecter des modèles de neurones humains et murins présentant des formes FAD. L’ensemble de cette démarche a permis d’ouvrir une nouvelle avenue à une potentielle thérapie pour la maladie d’Alzheimer.
Alzheimer’s disease (AD) is the most common form of dementia in the world, withnearly fifty million people affected currently. The most common symptoms of this diseaseare memory loss, difficulties in task management, and temporal and spatial confusions. There is currently no treatment for this disease. The amyloid precursor protein (APP) is usually cut by the alpha-secretase enzyme; however, abnormal cleavage by the beta-site APP cleaving enzyme 1 (BACE1) leads to the formation of beta-amyloid peptides. These peptides in turn forms aggregates, which accumulate as plaques in the brains of Alzheimer patients. Many non-silent APP mutationscause changes to the amino acid composition of the protein and result in increased plaque accumulation. These mutations are called familial forms of Alzheimer’s disease (FAD).However, one of these mutations (Icelandic A673T mutation) has been shown to confer aprotection against the on set and development of AD. This mutation of a single mutation inexon 16 changes an alanine into a threonine and has been shown to reduce the cleavage ofthe APP protein by BACE1 by 40%.This kind of single point mutation is the perfect target for the newly discoveredCRISPR/Cas9 technology, which opens new perspectives for the development of preventiveor curative treatments for genetic diseases and in our case Alzheimer’s. The Cas9endonuclease is a powerful tool for the modification of genetic data. The protein has been shown to cut double-stranded DNA with the help of a guide RNA (gRNA) to target a specified sequence adjacent to a PAM (protospacer adjacent motif). The base CRISPRsystem has been coopted by many different research teams; one of which used the technology to develop a technique they called base editing. This technique allows researchers toexchange cytidine bases for thymine and guanine bases for adenine with a strong accuracy. The first article of this thesis aims to demonstrate that the addition of the A673Tmutation in codominance with another pathological form of AD may have beneficial effectson the reduction of beta-amyloid peptides in patients’ brains. To determine if the mutationwas protective, plasmids carrying the A673T mutation along with another random FADmutation were used. Ultimately, we confirmed the beneficial effect for many forms of FAD,in particular the London V717I mutation demonstrated the greatest reduction in beta amyloidproteins. The second article of this thesis deals with the insertion of the A673T mutation by theCRISPR/Cas9 derived system, base editing. Several base editor complexes were compared and optimized to achieve the most effective and accurate genome modification possible. A candidate was selected after testing on HEK293T cells and SH-SY5Y neuroblastoma. The third part of this manuscript presents the results obtained when using lentiviraland AAV vectors to infect induced human and mouse neurons with a base editor complex and harvested mouse neurons with FAD forms. This whole approach has opened up an avenue for a potential therapy for Alzheimer’sdisease.

Les maladies à triplet sont dues à des expansions de trinucléotides dans l’ADN. Aucun traitement n’existe pour les soigner. Le but de cette thèse est de mettre au point de nouvelles approches de thérapie génique pour supprimer les expansions pathologiques dans le génome humain. Dans une première partie, un système expérimental dans la levure a été construit afin d’évaluer l’efficacité de différentes nucléases associées au système CRISPR sur des microsatellites. La seconde partie est concentrée sur une maladie à triplet en particulier ; la dystrophie myotonique de type 1 (DM1), qui est due à une expansion d’une répétition de triplets CTG dans la région 3’UTR du gène DMPK. Une nucléase, TALENCTG , construite pour induire une cassure double-brin dans les répétitions CTG en 3’UTR du gène DMPK, induit de manière très efficace des contractions de triplets CTG dans la levure. Des événements de contraction ont été observés lorsque cette nucléase est exprimée. Des expériences in vivo dans un modèle de souris contenant un fragment d’ADN génomique humain de patient contenant 1000 CTG ont été menées. Des particules virales AAV recombinantes portant le gène de la TALEN ont été produites. Après injection intramusculaire, les cellules musculaires expriment la nucléase, mais dû à une toxicité ou immunogénicité de la protéine, l’expression est perdue. Enfin, le système mis au point dans la levure a été transposé dans une lignée cellulaire humaine établie, les HEK293FS. Ce système pourra servir à sélectionner des nucléases actives dans les cellules humaines
Microsatellite disorders are a specific class of human diseases that are due to the expansion of repeated sequences above pathological thresholds. These disorders have varying symptoms and pathogenic mechanisms, caused by the expanded repeat. No cure exists for any of these dramatic conditions. This thesis is investigating new gene editing approaches to remove pathological expansions in the human genome. In a first part, a yeast-based screen was constructed to identify potent CRISPR-associated nucleases that can cut these microsatellites. The second part focuses on myotonic dystrophy type 1 (DM1), which is due to and expanded CTG repeat tract located at the 3’UTR of the DMKP gene. A nuclease, TALENCTG was designed to induce a double strand break into the CTG repeats. It was previously shown to be active in yeast cells, inducing contractions of CTG repeats from a DM1 patient integrated into the yeast genome. The TALEN was tested in DM1 patient cells. The nuclease was found to trigger some contraction events in patient cells. In vivo experiments were carried out in a mouse model of myotonic dystrophy type 1 containing a human genomic fragment from a patient and 1000 CTG. Intramuscular injections of recombinant AAV encoding the TALENCTG revealed that the nuclease is toxic and/or immunogenic in muscle cells in the tested experimental conditions. Finally, the reporter assay integrated in yeast to screen nucleases was transposed in HEK293FS cell line. The integrated cassette contains a CTG expansion from a myotonic dystrophy type 1 patient flanked by two halves of GFP genes. This system would enable to find nucleases active in human cells

La thérapie génique est une stratégie thérapeutique prometteuse pour le traitement des maladies monogéniques. Si les premières approches, dites additives, ont reposées sur l’utilisation de vecteurs viraux, une part grandissante se tourne désormais vers l’édition génique. Celle-ci est permise par la mise au point de nouvelles générations d’endonucléases, et en particulier le système CRISPR-Cas9. Moins d’une décennie après sa caractérisation, le système CRISPR-Cas9 a permis de faire passer l’édition génique à un stade clinique. Toutefois, dans le même laps de temps, plusieurs interrogations ont été soulevées vis-à-vis de la génotoxicité pouvant être induite par la Cas9. Une littérature émergente pointe le risque de génotoxicité au site ciblé. Le travail de thèse présentée ici s’inscrit dans cette thématique. La première partie de l’étude a eu pour objectif de décrire la génotoxicité induite par une unique cassure double-brin faite par la Cas9. La caractérisation des effets a été faite à la fois à l’échelle nucléotidique, par le suivi de la balance HDR / InDels, mais également à l’échelle du chromosome. Le suivi de l’intégrité chromosomique a permis de mettre en lumière un nouveau risque de génotoxicité encore non-caractérisé. Un système de détection sensible et spécifique de ce risque a été mis au point pour continuer de le caractériser. Le second objectif a été de répondre aux limites soulevées par la génotoxicité non-voulus, en mettant au point une méthode d’édition génique plus sûre et aussi efficace, via l’utilisation d’une unique cassure simple-brin par la Cas9D10A -nickase
Gene therapy is a promising therapeutic strategy for the monogenic diseases treatment. If the first approaches, called additive, have relied on the use of viral vectors, a growing share is now turning to gene editing. Less than a decade after its characterization, the CRISPR-Cas9 system has moved gene editing to a clinical stage. However, in the same period of time, several questions have been raised regarding the genotoxicity that can be induced by Cas9. An emerging literature points to the risk of genotoxicity at the targeted site. The thesis work presented here is part of this theme. The first part of the study aimed to describe the genotoxicity induced by a single double-stranded break made by Cas9. Characterization of the effects was done both at the nucleotide level, by monitoring the HDR / InDels balance, but also at the chromosome scale. The monitoring of chromosomal integrity has brought to light a new risk of genotoxicity that was not characterized. A sensitive and specific detection system for this risk has been developed to further characterize it. The second objective was to address the limitations of unwanted genotoxicity by developing a safer and more efficient gene editing method through the use of a single single-stranded breakage by Cas9D10A-nickase

Depuis quelques années, le poisson zèbre (Danio rerio) est devenu un modèle de choix pour l'étude du système nerveux et de ses fonctions. Récemment, des technologies nouvelles d'édition du génome permettent la génération d'allèles mutés de manière constitutionnelle et l'étude fonctionnelle de gènes chez ce modèle vertébré. Néanmoins, certains loci nécessite une inactivation spatiotemporelle précise et contrôlée. La première partie de ma thèse décrit la mise au point d'une nouvelle stratégie de disruption génétique de manière tissu-spécifique, basée sur la technologie du CRISPR/Cas9 et du système UAS/Gal4. Cette technique permet l'introduction de mutations somatiques dans des tissus, des clones ou des cellules individuelles préalablement génétiquement marqués, rendant ainsi possible le suivi in vivo de l'effet de la mutation générée grâce au gène rapporteur. La seconde partie de ma thèse se centre sur l'étude fonctionnelle d'une famille des gènes, les meteorines, durant le développement du système nerveux et lors du ciblage axonale chez le poisson zèbre. Les Meteorines sont des protéines conservées chez les vertébrés qui ont été impliquées dans la prolifération, la différentiation des progéniteurs de neurones et notamment dans l'élongation axonale in vitro. Nous avons pu mettre en évidence que les meteorines sont exprimées le long de la ligne médiane du système nerveux chez les larves et au niveau du plancher de la partie postérieure du cerveau et de la moelle épinière. Par l'utilisation du CRISPR/Cas9, nous avons généré des lignées mutantes pour chaque gène meteorine et avons ainsi procédé à l'analyse de l'établissement des projections axonales dans ces lignées mutantes
In recent years, the zebrafish (Danio rerio) has emerged as a powerful model organism to study neuronal circuit development and function. To date, different genome editing technologies allow the generation of constitutive mutant alleles, permitting the study of gene loss-of-function in this vertebrate model. Nevertheless, to assess the role of certain loci it might be required a precise spatiotemporal control of gene inactivation. The rst part of my thesis describes a novel strategy for tissue-specific gene disruption based on the CRISPR/Cas9 and the Gal4/UAS systems. The described technique allows the induction of somatic mutations in genetically labeled tissues, cell clones or single cells, making it possible to follow the effect of gene disruption in vivo via reporter gene expression. The second part of the thesis focuses on the functional analysis of the role of the meteorin gene family during neuronal development and axonal targeting in zebra sh. Meteorin family is conserved among vertebrates and its members have been shown to be involved in neuronal progenitor proliferation and differentiation and axonal elongation, in vitro. We used the zebrafish nervous system as a model to dissect the role of Meteorins during embryonic development, focusing on their potential role as novel guidance molecules. Interestingly, we found that genes belonging to the meteorin family are expressed along the midline of the larval central nervous system and at the floor plate in the hindbrain and spinal cord. We generated CRISPR/Cas9 mutant lines carrying out-of-frame deletions in the coding sequence of each member of the zebrafish meteorin family and we performed a comprehensive analysis of the establishment of axonal projections in the mutants. Our data pointed out that metrns loss-of-function affects the earliest process of axonal development, demonstrating a crucial role in the process of axonal outgrowth for this new family of evolutionary conserved guidance molecules

The CRISPR/Cas9 system has become the predominant tool for genome editing. Targeted modifications can be introduced while repairing double strand breaks (DSBs), induced by the CRISPR/Cas9 system. The DSB is repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR), and the repair is commonly processed through NHEJ because it is the dominant repair pathway in most cell types. The goal of this study is to modulate DNA repair system of somatic cells to increase the frequency of homology-directed repair (HDR) through HR by chemical treatment and the frequency of NHEJ by serum starvation. CRISPR/Cas9 systems targeting RAG2 gene and donor DNA to replace endogenous RAG2 were transfected into porcine fetal fibroblast (PFF) cells and the cells were treated with various chemicals that were known to inhibit NHEJ or stimulate HR. Among the chemical treated groups, cells treated with thymidine showed an average of 5.85-fold increase in HDR compared to the control group; the difference ranged from 1.37 to 9.59. There was no positive effect on the frequency of HDR after treating transfected cells with other chemicals. Placing PFFs under low amount of serum (serum deprivation) could enrich the cells in G0/G1 phase, but there was little difference in the frequency of NHEJ. Our results indicate that modulating DNA repair pathways during CRISPR/Cas9-mediated gene targeting could change the outcome of the targeted events.
Master of Science

Die Modifikation von Genen ist in den molekularen Biowissenschaften ein fundamentales Werkzeug, um die Funktion von Genen zu studieren (Reverse Genetik). Diese Arbeit hat erfolgreich Zinkfinger- und CRISPR/Cas9-Nukleasen für die Verwendung in C. reinhardtii etabliert, um Gene im Kerngenom gezielt auszuschalten und präzise zu verändern. Basierend auf vorausgegangener Arbeit mit Zinkfingernukleasen (ZFN) konnte die Transformationseffizienz um das 300-fache verbessert werden, was die Inaktivierung von Genen auch in motilen Wildtyp-Zellen ermöglichte. Damit war es möglich, die Gene für das Kanalrhodopsin-1 (ChR1), Kanalrhodopsin-2 (ChR2) und das Chlamyopsin-1/2-Gen (COP1/2) einzeln und gemeinsam auszuschalten. Eine Analyse der Phototaxis in diesen Stämmen ergab, dass die Phototaxis durch Inaktivierung von ChR1 stärker beeinträchtigt ist als durch Inaktivierung von ChR2. Um das CRISPR/Cas9-System zu verwenden, wurden die Transformationsbedingungen so angepasst und optimiert, dass der Cas9-gRNA-Komplex als in vitro hergestelltes Ribonukleoprotein in die Zellen transformiert wurde. Um die Bedingungen für präzise Genmodifikationen zu messen und zu verbessern, wurde das SNRK2.2-Gen als Reportergen für eine „Blau-Grün Test“ etabliert. Kleine Insertionen von bis zu 30 bp konnten mit kurzen Oligonukleotiden eingefügt werden, während größere Reportergene (mVenus, SNAP-Tag) mithilfe eines Donor-Plasmids generiert wurden. In dieser Arbeit konnten mehr als 20 nicht-selektierbare Gene – darunter 10 der 15 potenziellen Photorezeptorgene – mit einer durchschnittlichen Mutationsrate von 12,1 % inaktiviert werden. Insgesamt zeigt diese Arbeit in umfassender Weise, wie Gen-Inaktivierungen und Modifikationen mithilfe von ZFNs und des CRISPR/Cas9-Systems in der Grünalge C. reinhardtii durchgeführt werden können. Außerdem bietet die Sammlung der zehn Photorezeptor-Knockouts eine aussichtsreiche Grundlage, um die Vielfalt der Photorezeptoren in C. reinhardtii zu erforschen.
Gene editing is a fundamental tool in molecular biosciences in order to study the function of genes (reverse genetics). This study established zinc-finger and CRISPR/Cas9 nucleases for gene editing to target and inactivate the photoreceptor genes in C. reinhardtii. In continuation of previous work with designer zinc-finger nucleases (ZFN), the transformation efficiency could be improved 300-fold, which enabled the inactivation of genes in motile wild type cells. This made it possible to disrupt the Channelrhodopsin-1 (ChR1), Channelrhodopsin-2 (ChR2) and Chlamyopsin-1/2 (COP1/2) genes individually and in parallel. Phototaxis experiments in these strains revealed that the inactivation of ChR1 had a greater effect on phototaxis than the inactivation of ChR2. To apply the CRISPR/Cas9 system, the transformation conditions were adapted and optimized so that the Cas9-gRNA complex was successfully electroporated into the cells as an in vitro synthesized ribonucleoprotein. This approach enabled gene inactivations with CRISPR/Cas9 in C. reinhardtii. In order to measure and improve the conditions for precise gene modifications, the SNRK2.2 gene was established as a reporter gene for a ‘Blue-Green test’. Small insertions of up to 30 bp were inserted using short oligonucleotides, while larger reporter genes (mVenus, SNAP-tag) were integrated using donor plasmids. Throughout this study, more than 20 non-selectable genes were disrupted, including 10 of the photoreceptor genes, with an average mutation rate of 12,1 %. Overall, this work shows in a comprehensive way how gene inactivations and modifications can be performed in green alga C. reinhardtii using ZFNs or CRISPR/Cas9. In addition, the collection of the ten photoreceptor knockouts provides a promising source to investigate the diversity of photoreceptor genes in C. reinhardtii.

Cette thèse examine les mécanismes de l'immunité adaptative CRISPR-Cas chez les procaryotes, en utilisant principalement le système de type I-E chez Escherichia coli, en se concentrant sur le processus d'acquisition de spacers et la spécificité du système. Elle éclaire la dynamique de génération de spacers et leur intégration dans les CRISPR-arrays, en comparant les modes d'adaptation naïve et primed. L'étude révèle qu'une séquence proximale à un PAM (protospacer adjacent motif) particulier entrave l'acquisition de spacers en mode primed, fournissant un identifiant distinct pour les spacers acquis naturellement. L'étude révèle en outre le rôle des enzymes non-Cas, liées aux voies de réparation de l'ADN, dans la génération et le traitement des spacers, contribuant à l'adaptation et à l'interférence du CRISPR.Un autre volet de l'étude identifie les dangers potentiels posés par les effets hors cible causés par l'enzyme guidée par l'ARN Cas9 (dCas9) qui peut inadvertamment réduire au silence les gènes. Cela se produit lorsqu'il y a une correspondance minimale de seulement quatre nucléotides entre l'ARNg et la cible à l'intérieur de la séquence proximale du PAM, soulignant la nécessité d'une conception expérimentale soigneuse dans la recherche CRISPR-Cas. Dans l'ensemble, la thèse élargit la compréhension des mécanismes moléculaires complexes derrière l'adaptation du CRISPR, soulignant le rôle des protéines non-Cas et l'importance d'un contexte génétique spécifique des séquences proximales à un PAM, conduisant au développement d'outils de génie génétique plus précis et efficaces
This thesis investigates the mechanisms of the CRISPR-Cas adaptive immunity in prokaryotes, primarily using the type I-E system in Escherichia coli, focusing on the spacer acquisition process and the system's specificity. It sheds light on the dynamics of spacers generation and integration into CRISPR arrays, comparing naive and primed adaptation modes. The study reveals that a particular PAM (protospacer adjacent motif) - proximal sequence impedes spacer acquisition in the primed mode, providing a distinct identifier for naturally acquired spacers. The study further reveals the role of non-Cas enzymes, linked to DNA repair pathways, in spacer generation and processing, contributing to CRISPR adaptation and interference.Another course of the study identifies potential hazards posed by off-target effects caused by Cas9 (dCas9) RNA-guided enzyme that can inadvertently silence genes. This occurs when there's a minimal match of just four nucleotides between the gRNA and the target within the PAM-proximal sequence, emphasizing the need for careful experimental design in CRISPR-Cas research.Overall, the thesis expands understanding of the complex molecular mechanisms behind CRISPR adaptation, highlighting the role of non-Cas proteins and the significance of a specific genetic context of seed sequences, leading to the development of more precise and efficient genetic engineering tools

Hypertrophic cardiomyopathy (HCM) is a prevalent genetic cardiovascular disease affecting 1:500 individuals whose cardiac function is deteriorated due to thickening of the left ventricle of the heart, mostly owing to mutations in sarcomeric genes. Modeling HCM in vitro using human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) offers promise to further investigate the disease mechanisms, towards the development of effective drugs. Herein, nickase CRISPR/Cas9 genome editing technology was harnessed to introduce the R453C pathological mutation in the MYH7 sarcomeric gene, in three healthy hPSC lines. Monoclonal hPSC lines generated displayed the mutation in one or both alleles, as confirmed by PCR-genotyping and Sanger sequencing. A monolayer cardiac differentiation protocol was applied to the generated hPSC lines, resulting in >90% cardiomyocyte purities, and expression of mutant allele(s) of the MYH7 gene was analysed by RT-PCR. High-content imaging analysis showed that mutant hPSC-CMs displayed higher expression of hypertrophic marker Brain Natriuretic Peptide (BNP), in comparison to isogenic controls. BNP expression was maximised by treatment with hypertrophic inducer Endothelin-1 and rescued by its antagonist Bosentan. Flow cytometry analysis revealed a mild increase in cell volume of mutant cardiomyocytes relative to their wild-type controls. Functional evaluation of gene-edited lines exposed higher mitochondrial respiration rates relative to the isogenic controls, with the same mitochondrial content, resulting in a trend towards oxidative stress. Further genome engineering to incorporate a calcium indicator in R453C-MYH7 lines enabled confocal line analysis of calcium transients. MYH7-mutant hPSC-CMs exhibited higher frequency of irregular events in comparison to the healthy control, faster calcium kinetics, and higher resting cytosolic calcium concentration. Integration of hPSC-CMs in Engineered Heart Tissues (EHTs) and subsequent analysis of contractile force showed that mutant lines had a hypo-contractile and negative clinotropic phenotype relative to their isogenic controls. Moreover, R453C-MYH7 hEHTs showed a more pronounced negative force-frequency relationship in comparison with the healthy lines. These phenotypes were not rescued by treatment with cardiac myosin activator Omecamtiv Mecarbil, suggesting that targeting other mechanisms indirectly related with the contractile apparatus may be a preferred route to attenuate the observed pathological changes. Finally, transcriptomic analysis of gene-edited lines showed up-regulation of genes associated with fetal gene program, hypertrophy, fibrosis, apoptosis and autophagy, indicating potential molecular mechanisms associated with the observed phenotypes and HCM progression. Overall, hPSC-CMs bearing the R453C-MYH7 mutation exhibit the main molecular and functional hallmarks of HCM, providing a physiologically-relevant platform that enables further dissection of disease mechanisms and promotes pharmacological intervention.

Mutations in the SOD1 gene are the best characterized genetic cause of amyotrophic lateral sclerosis (ALS) and account for ~20% of inherited cases and 1-3% of sporadic cases. The gene-editing tool Cas9 can silence mutant genes that cause disease, but effective delivery of CRISPR-Cas9 to the central nervous system (CNS) remains challenging. Here, I developed strategies using canonical Streptococcus pyogenes Cas9 to silence SOD1. In the first strategy, I demonstrate effectiveness of systemic delivery of guide RNA targeting SOD1 to the CNS in a transgenic mouse model expressing human mutant SOD1 and Cas9. Silencing was observed in both the brain and the spinal cord. In the second strategy, I demonstrate the effectiveness of delivering both guide RNA and Cas9 via two AAVs into the ventricles of the brain of SOD1G93A mice. Silencing was observed in the brain and in motor neurons within the spinal cord. For both strategies, treated mice had prolonged survival when compared to controls. Treated mice also had improvements in grip strength and rotarod function. For ICV treated mice, we detected a benefit of SOD1 silencing using net axonal transport assays, a novel method to detect motor neuron function in mice before onset of motor symptoms. These studies demonstrate that Cas9-mediated genome editing can mediate disease gene silencing in motor neurons and warrants further development for use as a therapeutic intervention for SOD1-linked ALS patients.

Modelos humanizados de porco são muito importantes para pesquisa biomédica e desenvolvimento de novas drogas e tratamentos. Além de ser um melhor modelo para doenças humanas do que animais de menor porte devido sua maior semelhança fisiológica, anatômica, de metabolismo e tempo de vida, o modelo suíno ainda permite suprimento ilimitado de órgãos para transplante. Apesar dessas vantagens, a expressão gênica inconsistente de animais transgênicos tornam a criação e avaliação desses animais muito dispendiosas, imprevisível e não permite a comparação de resultados de animais diferentes de maneira apropriada. Nesse estudo descrevemos uma nova técnica utilizando o promoter endógeno para a geração de um protocolo de substituição de genes com padrão clonal (transplante clonal de genes) sem clonagem de células, preservando a expressão genética e sua regulação intactas. Esse protocolo é reprodutível e pode ser aplicado para mais de um alvo genético, permitindo geração rápida de linhas transgênicas de animais (14-20 dias) com potencial de se tornar o novo padrão para geração de animais transgênicos de grande porte Suínos
Humanized pig models are very important for biomedical research, and drugs and treatment development. Not only it is a better model for diseases than smaller animals because of its closer physiology, anatomy, metabolism and life span, it also may provide unlimited organs for transplantation. In spite of all this advantages, inconsistent gene expression in transgenic animals make its generation and evaluation expensive, unpredictable and do not allow proper outcome comparison between different animals. In this report we describe a reproducible technique utilizing the endogenous promoter for generation of a clonal pattern gene replacement protocol (clonal gene transplant) without cell cloning, maintaining the normal gene expression and its regulation. This protocol is reproducible and applicable to more than one gene target, allowing fast generation of transgenic animals cell lines (as low as 14-20 days) and could become the new standard for transgenic large animal generation

The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23-and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. major HSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR–Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite’s biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.
Alexander von Humboldt-Stiftung
Revisión por pares

La salinité de l'eau d'irrigation et des sols est l'une des principales contraintes abiotiques en agriculture. Parmi les 130 mha de riz cultivés dans le monde, 30% environ se trouvent dans des zones où la salinité est trop élevée pour permettre de bons rendements. Dans l'objectif de mieux comprendre le rôle des systèmes de transport de Na+ dans la compartimentation de Na+ dans la plante permettant de maintenir un rapport K+/Na+ élevé dans les tissus sensibles lors d’un stress salin, je me suis intéressé à deux transporteurs de la famille HKT, OsHKT1;1 et OsHKT1;3, chez le riz, la plante modèle des céréales. Mon principal projet a allié une stratégie "promoteur::GUS" pour préciser les patrons d'expression in planta des deux gènes, et une approche de génétique inverse en produisant des mutants pertes de fonction à l’aide de la technologie CRISPR-Cas9, qui s’est confirmée être un outil efficace pour générer des mutations indèles après cassure ciblée de l’ADN, transmissibles de façon stable chez le riz. Les tests histochimiques de l’activité GUS ont montré une expression prédominante d’OsHKT1;1 et d’OsHKT1;3 dans les tissus vasculaires (parenchyme xylémien et/ou phloème), essentiellement au niveau des parties aériennes. L’analyse phénotypique des plantes "CRISPR" a mis en évidence des modifications des profils d’accumulation de Na+ dans les feuilles chez les mutants: défaut d’accumulation de Na+ dans les gaines foliaires pour les plantes oshkt1;1 et répartition altérée de Na+ entre les vieilles et les jeunes feuilles chez les plantes oshkt1;3, conduisant à une augmentation de la teneur en Na+ dans les limbes des jeunes feuilles chez les 2 types de mutants. Dans l’ensemble, ces résultats suggèrent qu’OsHKT1;1 et OsHKT1;3 participent au dessalage des jeunes limbes foliaires lors d’un stress salin, via des mécanismes différents, contrôlant les transports xylémiques et phloémiens de Na+. En plus de cette étude, j’ai collaboré avec un groupe de généticiens, qui avait identifié une forte association entre des différences écotypiques de contenu racinaire en Na+ et de rapport K+/Na+, et une région du chromosome 4 du riz comprenant le gène OsHKT1;1. En comparant fonctionnellement par électrophysiologie les deux variants majeurs d’OsHKT1;1, des différences de capacités de transport de Na+ susceptibles d’expliquer le trait racinaire ont été observées. L’ensemble des données obtenues souligne l’intérêt d’analyser tous des gènes HKT de riz dans les mécanismes de tolérance de la plante à la salinité
The salinity of irrigation water and soils is one of the main abiotic constraints in agriculture. Among the 130 mha of rice grown around the world, about 30% are in areas where salinity is too high to allow for good yields. In order to better understand the role of Na+ transport systems in the compartmentalization of Na+ in the plant to maintain a high K+/Na+ ratio in sensitive tissues during salt stress, I focused on two transporters from the HKT family, OsHKT1;1 and OsHKT1;3, in rice, the model cereal species. My main project combined a “promoter::GUS” strategy to specify the expression pattern of both genes, and a reverse genetics approach by producing loss-of-function mutants using the CRISPR-CAS9 technology, which was confirmed to be an efficient tool for generating indel mutations after targeted DNA breaks, stably transmitted in rice. Histochemical tests of GUS activity showed predominant expression of OsHKT1;1 and OsHKT1;3 in vascular tissues (xylem parenchyma and/or phloem), mainly in the aerial parts. Phenotypic analysis of "CRISPR" plants revealed changes in leaf Na+ accumulation profiles in mutants: lack of Na+ accumulation in leaf sheaths for oshkt1;1 plants and altered distribution of Na+ between old and young leaves in oshkt1;3 plants, leading to an increase in the leaf blade Na+ content in both types of mutants. Overall, these results suggest that OsHKT1 and OsHKT1.3 contribute to the desalination of young leaf blades during salt stress, via different mechanisms, controlling transports of Na+ through the xylem and phloem. In addition to this study, I collaborated with a group of geneticists who identified a strong association between ecotypic differences in root Na+ and K+/Na+ ratio, and a region of chromosome 4 of rice including the OsHKT1;1 gene. By comparing electrophysiologically the two major variants of OsHKT1;1, differences in Na+ transport capacities that could explain the root trait were observed. All the data obtained underline the interest of analyzing all HKT genes of rice in the mechanisms of plant tolerance to salinity

Malaria is a parasitic disease caused by parasites of the Plasmodium genus and in 2015 around 214,000,000 new cases of malaria infection occurred resulting in 438,000 deaths worldwide. Currently, there is no available vaccine against malaria and despite efforts, mainly channelled into subunit vaccines, immunogenicity is often low. In this work, whole live blood stage Plasmodium was used in oral and nasal inoculation of mice to test for infectivity and immunogenicity. Oral inoculation resulted in infection of some mice while none of those inoculated nasally became infected. Both oral and nasal inoculation, despite not providing protection, allowed for extended survival of inoculated individuals. Given that survival was increased by inoculating animals solely with live parasites, the results detailed in this work show that this approach holds great applicational potential, as coupling oral and nasal inoculation with live parasites and a powerful adjuvant may be the key to unlock full protection. In connection with inoculation of live parasites, this work also aimed at producing a Plasmodium falciparum mutant expressing Salmonella’s FimH+ glycoprotein as to allow its detection by M cells in the gut. This was to be achieved by a modified version of the CRISPR/Cas9 system which would bear a double guide RNA instead of a single. However, this was not possible as the resulting CRISPR plasmid contained an extremely high AT-content becoming toxic to E. coli. In addition to these two main goals, a few molecular techniques crucial for malaria research were improved and a detection method of avian malaria was developed.

La dystrophie Facio-Scapulo-Humérale (FSHD) fait partie des maladies musculaires génétiques les plus fréquentes. Elle se caractérise par une dégénérescence progressive et asymétrique d’un groupe spécifique de muscles striés squelettiques, dont principalement les muscles faciaux, scapulaires et huméraux. D’un point de vue génétique, la FSHD est une maladie multifactorielle qui résulte d’évènements génétiques situés sur la région sub-télomérique du chromosome 4, ainsi que d’évènements épigénétiques altérant l’organisation chromatinienne du locus 4q35. Ces anomalies provoquent une relaxation chromatinienne et une surexpression de la majorité des gènes du locus 4q35, dont DUX4, gène majeur impliqué dans la FSHD. Les répercussions de l’ensemble de ces altérations se traduisent notamment par une dérégulation de la signature transcriptionnelle des myoblastes primaires issus des patients FSHD, et par des anomalies de leur différenciation myogénique in vitro et leur hypersensibilité au stress oxydant. Plusieurs aspects de la maladie demeurent incompris, et la complexité de cette myopathie rend difficile le choix d’une stratégie thérapeutique optimale. Cependant, la découverte des outils de l’édition du génome et la multiplication de leurs applications à visée thérapeutique dans le cadre de maladies humaines, notamment les myopathies, ouvre de nouvelles perspectives pour la FSHD qui reste, jusque-là, incurable.Le travail de thèse a concerné, dans un premier temps, l’implication des dommages de l’ADN et du stress oxydant dans la pathophysiologie de la FSHD. Nous avons mis en évidence l’omniprésence de ces caractéristiques cellulaires dans les myoblastes FSHD, leur lien à l’expression aberrante de DUX4 et leur participation à la morphologie défectueuse des myotubes FSHD in vitro. Dans un second temps, le travail de thèse a consisté à concevoir et à développer des outils de l’édition génomique et épigénomique, capables de cibler spécifiquement un des évènements génétiques causal de la FSHD, le variant pathogénique 4qA161 touchant un site d’attachement à la matrice nucléaire, FR-MAR. A partir de ces outils développés, deux stratégies de corrections génomique et épigénomique à visée thérapeutique peuvent être alors envisagées in vitro, ayant pour but ultime de rétablir la fonction d’insulation de FR-MAR et la conformation chromatinienne de la région 4q35
Facioscapulohumeral dystrophy (FSHD) is one of the most common genetic myopathies characterized by a progressive and asymmetric weakening of a specific group of skeletal muscles, typically facial, shoulder girdle and upper arms muscles. FSHD is a multifactorial disease that results from the combination of genetic and epigenetic events mapped at the 4q35 locus. These genetic and epigenetic alterations lead to chromatin relaxation and the subsequent overexpression of the majority of 4q35 genes, notably DUX4, the major actor in FSHD pathology. These genomic alterations lead to molecular and cellular defects observed in vitro. Cultured-FSHD myoblasts show a distinct transcription profile, they exhibit morphological differentiation defects and are sensitive to oxidative stress. Several aspects of the disease remain poorly understood, and the elaboration of an appropriate therapeutic strategy is limited by the complexity of this myopathy. However, the discovery of genome editing tools and their successful therapeutic applications in vitro and in animal models of several human diseases, including myopathies, open doors to potential therapeutic strategies for FSHD.This work highlighted the involvement of DNA damage and oxidative stress in the pathophysiology of FSHD, by revealing their constitutive presence in FSHD myoblasts, their link to DUX4 expression and their participation in morphological defects of FSHD myotubes observed in vitro. The second part of this work was aimed at developing genome- and epigenome-editing tools capable of specifically targeting one of the genetic events causing FSHD, a pathogenic variant 4qA161 that contains an insulator and a nuclear matrix attachment site (FR-MAR). These engineered tools will be then used to develop in vitro therapeutic strategies, with the intention of restoring the insulator activity of FR-MAR and the chromatin organization of 4q35 locus

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第22503号
農博第2407号
新制||農||1077(附属図書館)
学位論文||R2||N5283(農学部図書室)
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 佐藤 健司, 教授 澤山 茂樹, 准教授 豊原 治彦
学位規則第4条第1項該当

Mitochondria are very dynamic organelles with a crucial role in life and death of eukaryotic cells. These organelles regulate cellular energy generation, calcium and redox homeostasis, and apoptosis. To perform the cellular functions effectively, mitochondria continuously change their structure and morphology through protein machineries controlling fission and fusion process (mitochondrial dynamics). Strong evidence has emerged to implicate disturbed mitochondrial fusion and fission as central pathological components underpinning a number of childhood and adult-onset neurodegenerative disorders. Several proteins that regulate the morphology of the mitochondrial network have been identified, the most widely studied of which are Optic Atrophy 1 (OPA1), Mitofusin1 and 2 (Mfn1 and 2) and Dynamin Related Protein 1 (DRP1). OPA1 is a ubiquitously expressed dynamin-like GTPase in the inner mitochondrial membrane. It plays important roles in mitochondrial fusion, apoptosis, reactive oxygen species (ROS) and ATP production. Mutations of OPA1 result in autosomal Dominant Optic Atrophy (DOA), a common hereditary optic neuropathy characterized by retinal ganglion cell degeneration leading to optic neuropathy, symmetrical central visual loss and dyschromatopsia. The majority of OPA1 mutations result in premature termination codons, and the resultant truncated mRNA species are highly unstable, being rapidly degraded by protective surveillance mechanisms operating via nonsense-mediated mRNA decay. Haploinsufficiency, therefore, is a major disease mechanism in DOA, and the pathological consequences of a dramatic reduction in OPA1 protein levels is highlighted by those rare families who are heterozygous for microdeletions spanning the entire OPA1 coding region. Progressive visual failure remains the defining feature of DOA but, with greater availability of genetic testing, a specific OPA1 mutation in exon 14 (c.1334G>A, p.Arg445His) has been found to cause sensorineural deafness, ataxia, myopathy, peripheral neuropathy, and classical chronic progressive external ophthalmoplegia. This syndrome is called DOA plus. The molecular mechanisms linking OPA1 mutations and DOA are not fully understood. In this work a new model of OPA1-linked Dominant Optic Atrophy was generated in Drosophila melanogaster, in order to use it for studying DOA pathogenesis. The Drosophila OPA1 gene (dOPA1) shares 51.2% similarity with its human orthologue and the alignment of protein sequence of hOPA1 with dOPA1 shows that the domains most subjected to pathogenic mutations are well conserved. To address the pathophysiological mechanism of OPA1-linked DOA, we generated two dOPA1 mutants: OPA1 R417H, a mutant that carries in endogenous dOPA1 the mutation corresponding to R445H in humans; OPA1null carrying a microdeletion leading to production of a inactive truncated protein of 482 amino acids. To model these mutations we have used in vivo CRISPR/Cas9, a genome engineering system that has revolutionized genetic analysis in many organisms. For use in genome engineering the system requires two essential components: gRNA and Cas9-endonuclease. The gRNA recognizes a 20-nt target sequence next to a trinucleotide NGG protospacer adjacent motif (PAM) to direct Cas9-dependent cleavage of both DNA strands within the target sequence. Several groups have used the CRISPR/Cas9 system to induce targeted mutations in Drosophila, but differ in their approach to supply the Cas9 protein and gRNA components of the system. It has been demonstrated that two targeting gRNAs can be used to generated a large defined deletions and the Cas9 catalyzed gene replacement by homologous recombination. The experimental design of my work requires the following steps: generation of the gRNAs responsible for precisely targeting the genomic region where recombination should take place; generation of the dsDNA templates containing the desired genomic modifications to be introduced and homology arms for accurate recombination; choice of a screening method. The gRNAs guide the cut of the Cas9 on the genomic region of the dOPA1 gene through the target sequences and the PAM sites; the cut of genomic DNA favors homologous recombination with the dOPA1 mutated fragment cloned into dsDNA plasmid donor. The exogenous dOPA1 mutated gene in addition to the pathological mutations carries a silent mutation that introduces a novel BamHI restriction site necessary for screening the occurrence of homologous recombination events by restriction digest. The gRNAs and the dsDNA plasmids donors were microinjected in the embryo of the Drosophila line expressing Cas9 protein in the ovary under control of vasa regulatory sequences. The mutants were screened for the presence of the mutation through PCR on genomic DNA, restriction digest and sequencing of the OPA1 mutated fragment. After having verified that the mutants had mutations of interest without any other alterations, we described the phenotypic effects observed in these mutants. We also analyzed the mitochondria in the nervous and muscular systems using confocal microscopy and the mitochondrial functions through biochemical assays. Observations of the adults within the lines shows that both mutations in heterozygosity do not cause any evident morphological alterations. However, both mutations in homozygosity turned out to be lethal but differently R417H homozygous mutants develop until the second instar larva stage whereas the OPA1null homozygotes die earlier at the first instar larva stage. We were more interested in studying the heterozygote dOPA1 mutants because DOA is a dominant disease. The lifespan reduction of both dOPA1 mutants indicate that the heterozygous mutations of OPA1 is likely to cause systemic consequences probably affecting multiple processes. Since OPA1 is involved in mitochondrial dynamics we performed a series of experiments to analyze mitochondrial morphology in the neuronal and muscular systems of both dOPA1 mutants. Heterozygous dOPA1 mutants display defects of mitochondria morphology in nerves and muscles in Drosophila third instar larvae, mitochondria network shape is characterized by mild fragmentation and clusterization. Mitochondria function was analyzed on homozygous and heterozygous dOPA1 mutants. Mitochondrial respiration and the redox activity of respiratory complexes was decreased in both mutants. Furthermore heterozygous OPA1 R417H displayed more severe effects in some assays than OPA1null heterozygotes. This suggested that R417H mutation could interfere with the activity of the wild type copy of dOPA1 resulting in more severe phenotypes than those caused by the presence of a single loss of function allele. In conclusion, we have produced a model to study the Dominant Optic Atrophy which can be helpful to understand the pathogenesis of this disease caused by different classes of mutations within the OPA1 gene.
I mitocondri sono organelli dinamici fondamentali per la vita e la morte delle cellule eucariotiche, svolgono diverse funzioni tra cui: produzione di ATP, regolazione dell’omeostasi del Ca2+, produzione di ROS e regolazione dell’apoptosi. I processi di fusione e fissione mitocondriale (dinamiche mitocondriali) sono alla base del corretto funzionamento di questi organelli e sono controllati da una serie di proteine che, di conseguenza, regolano forma e struttura dei mitocondri. Disturbi delle dinamiche mitocondriali sono alla base di diverse patologie neurodegenerative che colpiscono bambini e giovani adulti. Le diverse proteine che regolano la morfologia della network mitocondriale sono state individuate in vari studi, le principali sono: Optic Atrophy 1 (OPA1), Mitofusin1 and 2 (Mfn1 and 2) and Dynamin Related Protein 1 (DRP1). OPA1 è una proteina ubiquitaria della famiglia delle dianamine, con attività GTPasica, situata sulla membrana interna dei mitocondri. Presenta un ruolo fondamentale nel processo di fusione mitocondriale, apoptosi, produzione di ROS e produzione di ATP. Mutazioni di OPA1 sono alla base dell’ Atrofia Ottica Dominante (DOA), una comune neuropatia ottica ereditaria caratterizzata da degenerazione delle cellule ganglionari della retina con conseguente neuropatia, perdita della capacità visiva simmetrica centrale e discromatopsia. La maggior parte delle mutazioni patologiche di OPA1 determinano la formazione di codoni di stop, con conseguente produzione di forme troncate di mRNA altamente instabili che vengono rapidamente degradate dai vari meccanismi di controllo. L’aploinsufficienza è il principale meccanismo patogenetico della DOA, le conseguenze di una drastica riduzione dei livelli di OPA1 sono ben visibili in famiglie in cui sono state identificate microdelezioni in eterozigosi, localizzate nella regione codificante del gene di OPA1. La perdita progressiva della vista rimane la caratteristica principale della DOA ma, la maggior disponibilità di test genetici, ha permesso di identificare una mutazione specifica del gene OPA1 localizzata sull’esone 14 (c.1334G>A, p.Arg445His) che causa DOA, associa a sordità neurosensoriale, atassia, miopatia, neuropatia periferica e oftalmoplegia esterna progressiva cronica. Questa sindrome è chiamata DOA plus. I meccanismi molecolari alla base di DOA causata da mutazioni di OPA1 non sono del tutto chiari. In questo lavoro abbiamo generato un nuovo modello di Atrofia Ottica Dominante usando Drosophila melanogaster, al fine di utilizzarlo per studiare e comprendere al meglio la patogenesi di questa malattia. Il gene OPA1 di Drosophila (dOPA1) mostra il 51.2% di similarità con il gene ortologo umano, l’allineamento della proteina umana con quella di Drosophila mostra che i domini, su cui si localizzano la maggior parte delle mutazioni patologiche, sono altamente conservati. Per studiare il meccanismo patofisiologico della DOA dovuta a mutazioni di OPA1, abbiamo generato due mutanti dOPA1: OPA1 R417H, un mutante che porta la mutazione corrispondente alla mutazione umana OPA1 R445H; e il mutante OPA1null in cui è stata inserita una microdelezione che determina la produzione di una forma tronca inattiva di 482 aminoacidi. Per inserire queste mutazione nel genoma di Drosophila abbiamo utilizzato il sistema CRISPR/Cas9, un sistema che permette la modifica del DNA genomico e che ha rivoluzionato le analisi genetiche in diversi organismi. Le componenti fondamentali di questo sistema sono gRNA e endonucleasi Cas9. Il gRNA riconosce una sequenza target di 20nt seguita da un sito PAM (protospacer adjacent motif), costituito da tre nucleotidi NGG, e necessario per indirizzare il taglio dei due filamenti del DNA genomico ad opera dell’endonucleasi Cas9. Diversi gruppi di ricercatori hanno utilizzato il sistema CRISPR/Cas9 per introdurre specifiche mutazioni nel genoma di Drosophila, mettendo a punto vari metodi di somministrazione delle diverse componenti. È stato dimostrato che un approccio metodologico efficace, è l’utilizzo di una coppia di gRNA che, mediante il taglio del Ca9, determinano una larga delezione in una porzione genomica ben definita e questo, in presenza di una dsDNA donatore, favorisce la sostituzione genica mediante ricombinazione omologa. Il disegno sperimentale del mio lavoro richiede i seguenti steps: generazione dei gRNA in grado di indirizzare il taglio dell’endonucleasi sulla regione genomica di interesse; generazione del dsDNA donatore contenete la porzione genica con le mutazioni desiderate e due regioni di omologia limitrofe ad essa e necessarie per la corretta ricombinazione e infine la scelta di un metodo di screening. I gRNA guidano il taglio del Cas9 sulla porzione genomica del gene dOPA1 grazie alla sequenza target di 20nt e ai siti PAM; il taglio del DNA genomico favorisce la ricombinazione omologa con il frammento mutato di dOPA1 clonato nel plasmide dsDNA donatore. Sul frammento genico esogeno, oltre alla mutazione patologica di OPA1, è stata inserita una mutazione silente per introdurre un sito di restrizione dell’enzima BamHI, necessario per lo screening dei mutanti in cui è avvenuta la corretta ricombinazione. I gRNAs e i plasmidi dsDNA donatori sono stati microiniettati in embrioni di una linea di Drosophila in cui la proteina Cas9 è espressa nelle cellule germinali, sotto il controllo del fattore di regolazione della trascrizione genica, vasa. Lo screening per individuare i mutanti corretti è stato fatto mediante PCR sul DNA genomico, taglio di restrizione e sequenziamento. Dopo aver verificato la corretta ricombinazione omologa del frammento esogeno abbiamo eseguito una caratterizzazione fenotipica dei mutanti. Mediante microscopia confocale, abbiamo analizzato la morfologia dei mitocondri nel sistema nervoso e muscolare; con dei saggi biochimici abbiamo poi testato la funzionalità mitocondriale in larve mutanti. Gli adulti eterozigoti per le mutazioni di dOPA1 non presentano evidenti alterazioni morfologiche. Entrambe le mutazioni però, risultano letali in omozigosi ma con delle differenze: la mutazione R417H risulta essere letale al secondo stadio larvale mentre la completa assenza di OPA1, che si determina nel mutante omozigote OPA1null, è letale al primo stadio larvale. Essendo DOA una malattia genetica dominante, è importante studiare gli effetti di queste mutazioni sui mutanti eterozigoti. Analizzando la durata media della vita dei mutanti adulti eterozigoti, abbiamo osservato un importante riduzione di questo parametro, simile per entrambe le mutazioni e collegato probabilmente agli effetti sistemici che si hanno in seguito all’alterazione di vari processi cellulari che coinvolgono OPA1 e i mitocondri. Dato che OPA1 è una proteina coinvolta nella regolazione delle dinamiche mitocondriali, abbiamo messo a punto una serie di esperimenti per analizzare la morfologia mitocondriale nel sistema nervoso e muscolare dei mutanti dOPA1. In larve terzo stadio eterozigoti per entrambe le mutazioni abbiamo osservato alterazioni della morfologia mitocondriale in nervi e muscoli, il network mitocondriale è caratterizzato da lieve frammentazione e presenza di cluster. La funzionalità mitocondriale è stata analizzata nei mutanti dOPA1 eterozigoti ed omozigoti. Respirazione mitocondriale e attività redox dei complessi della catena respiratoria risultano ridotte in entrambi i mutanti. Inoltre, il mutante OPA1 R417H eterozigote risulta avere deficit di funzionalità mitocondriale maggiore rispetto al mutante OPA1null eterozigote. Questo suggerisce che la mutazione R417H possa interferire con l’attività della copia wild type di dOPA1, determinando un fenotipo più grave della perdita di un solo allele funzionante. Per concludere, possiamo affermare di aver prodotto un modello di Atrofia Ottica Dominate che potrebbe essere d’aiuto per lo studio della patogenesi di questa malattia e per comprendere meglio come agiscono le diverse classi di mutazioni sul gene OPA1

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.

MHCI molecules are constitutively expressed in all nucleated cells and play a key role in antigen presentation to CD8+ T lymphocytes. One of the tumor immune evasion strategies is MHCI expression downregulation. This leads to an impaired recognition of tumor antigens by CD8+ T lymphocytes that are unable to start the immune response. Since the MHCI expression downregulation occurs in up to 90 % of some tumors it is neccesary to have a clinical relevant tumor model without a MHCI surface expression that would be used for testing of immunotherapeutic approaches. This thesis describes a production of new model cell lines of TC-1 tumor cells with irreversibly downregulated MHCI. That was achieved by an inactivation of B2m, which is a part of MHCI, by gene editing using CRISR/Cas9. The B2m inactivation was confirmed by flow cytometry, western blot and sanger sequencing of single alleles. The inactivation slowed down the cell growth for both in vitro and in vivo. The cell metastatic activity was not affected. The tumors established by cells without the B2m expression are not sensitive to DNA vaccine against HPV16 E7 oncoprotein by a pBSC/PADRE.E7GGG vaccine. The main effector function against these tumors possess the NK1.1+ cells. In a therapeutic vaccination experiment it was repeatedly achieved of...

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
The Integrated Master's Degree in Pharmaceutical Sciences of the Faculty of Pharmacy at Coimbra's University, after nine semesters of study, ends with the achievement of the Curricular Internship and elaboration of the monography. The internships carried out at Farmácia Roldão and at Laboratórios Expanscience are described here under the SWOT (Strengths, Weaknesses, Opportunities and Threats) analysis structure and this document also contains the monography entitled "Lipid Nanoparticles as Delivery Systems for Macromolecules".In the context of diseases with low expectations in the solutions offered by conventional therapies, gene therapy has proven to be the light at the end of the tunnel by delivering macromolecules capable of silencing pathological genes or expressing therapeutic proteins or even macromolecules with genetic correction capacity.Due to the numerous limitations of viral vectors, non-viral vectors are gaining more notoriety for ensuring greater safety. One of the most promising delivery systems are Lipidic Nanoparticles (LNPs) as they are one of the non-viral vectors that have demonstrated to be suitable for the delivery of macromolecules such as nucleic acids and gene editing enzymes.This review begins with the characteristics that NPLs should exhibit, such as the challenges that this system faces to be considered a successful delivery system for transporting nucleic acids in its intact form.Two current therapeutic solutions using this technology (Onpattro and Sars-CoV-2 vaccines) are then presented, as well as other CRISPR/Cas9-related research currently under development.
O Mestrado Integrado em Ciências Farmacêuticas da Faculdade de Farmácia da Universidade de Coimbra, após nove semestres de estudo, encerra com a concretização do Estágio Curricular e elaboração da monografia. Os estágios realizados na Farmácia Roldão e nos Laboratórios Expanscience são aqui descritos sob a estrutura de análise SWOT (Strenghs, Weaknessess, Opportunities and Threats) e o presente documento alberga ainda a monografia intitulada “Nanopartículas Lipídicas como Sistemas de Entrega de Macromoléculas”.No contexto de doenças com poucas expetativas nas soluções que as terapias convencionais oferecem, a terapia génica tem demonstrado ser a luz ao fundo do túnel através da entrega de macromoléculas capazes de silenciar genes cuja expressão desregulada se traduz numa patologia ou expressar proteínas terapêuticas ou até macromoléculas com capacidade de correção genética. Devido às inúmeras limitações dos vetores virais, os vetores não virais ganham maior notoriedade por garantir uma maior segurança. Um dos sistemas de entrega mais promissores são as Nanopartículas Lipídicas (NPL) por serem dos vetores não virais que demonstram ser o indicado para a entrega de macromoléculas como ácidos nucleicos e complexos de enzimas de edição de genes. A presente revisão inicia-se com as características que as NPL deverão apresentar, como os desafios que este sistema enfrenta para ser considerado um sistema de entrega de sucesso no transporte de ácidos nucleicos de forma íntegra. São apresentadas então duas soluções terapêuticas atuais que recorrem a esta tecnologia (caso do Onpattro® e das vacinas contra o SARS-CoV-2), também como outras investigações, relacionadas com o CRISPR/Cas9, que estão a ser desenvolvidas no momento.

abstract: The portability of genetic tools from one organism to another is a cornerstone of synthetic biology. The shared biological language of DNA-to-RNA-to-protein allows for expression of polypeptide chains in phylogenetically distant organisms with little modification. The tools and contexts are diverse, ranging from catalytic RNAs in cell-free systems to bacterial proteins expressed in human cell lines, yet they exhibit an organizing principle: that genes and proteins may be treated as modular units that can be moved from their native organism to a novel one. However, protein behavior is always unpredictable; drop-in functionality is not guaranteed. My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli. 1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin. 2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
Dissertation/Thesis
Doctoral Dissertation Bioengineering 2017

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

Gene regulatory networks, underlying the molecular regulation of eye development are conserved across many animal phyla. Genes from the Pax family of transcription factors are one of the most conserved members through the evolution, regulating the development of crucial parts of eye, including the photoreceptor cells. Pax transcription factors are considered to be regulators of opsins, molecules providing the conversion of the light stimulus into the electrochemical signalisation in the photoreceptors cells. In this thesis, pax6 and pax2/5/8 transcription factors are investigated as potential regulators of eye development in Platynereis dumerilii. pax6 and pax2/5/8 transcription factors are tested as potential regulators of the r-opsin in Platynereis, based on the observed early expression onsets of these genes. Wild-type expression analysis of pax6 and pax2/5/8 using the whole mount RNA in-situ hybridization is provided, accompanied by the initial analysis of the Platynereis pax6 knockout line. pax6 heterozygote mutants are shown to be viable and able to reproduce, however, homozygote mutation of pax6 in Platynereis is lethal. Our data suggest that transcription factors pax2/5/8, otx and six3 are not regulated by the pax6 in Platynereis. Concerning the r-opsin present in the Platynereis eyes, pax6...

Dissertação de mestrado em Bioengenharia
Curcumin has been reported for its beneficial therapeutic properties including as anti-cancer agent. However, it has poor bioavailability and it is quickly metabolized in the human body, implying a repetitive oral administration if a therapeutic effect is envisaged. Besides, its extraction from plants is very expensive. For these reasons, the use of microorganisms to produce it on large scale and with greater yields constitutes an interesting alternative. With this aim, Escherichia coli K-12 MG1655 (DE3) was previously engineered with three enzymatic steps (4-coumarate-CoA ligase, diketide-CoA synthase and curcumin synthase 1) that catalyze the production of curcumin from ferulic acid. In the present study, the optimal strain, operational conditions and media composition for the production of curcumin by E. coli harboring the artificial biosynthetic pathway were established. Previously, a standard two-step fermentation strategy (LB+M9 minimal medium) was used. Although feasible at the laboratory scale, the biomass separation is much more difficult, laborious and expensive in large-scale fermentations. Therefore, herein a single medium formulation more suitable for the production of curcumin at an industrial set-up was implemented. MOPS minimal medium, TB and LB were evaluated. Using the optimized conditions, the curcumin concentration obtained in this study was the highest reported to be produced by a heterologous organism, 686.7±59.7 µM in TB (43 h) and 822.6±28.1 µM in LB+M9 (63 h). These results were obtained using E. coli BL21 (DE3) that was identified as the best producer since it produced 3.7 times more curcumin than E. coli K-12 MG1655 (DE3). Moreover, curcumin toxicity against E. coli cells was evaluated. The tests performed showed that curcumin concentrations above 400 µM influence negatively the E. coli cells growth. Furthermore, one of the purposes of the current work was to construct and validate several E. coli mutants (e.g. ΔfumA,fumB,fumC) previously identified by an in silico approach as the most promising towards an increased production of curcumin from ferulic acid. The deletion of fumB gene from E. coli K-12 MG1655 (DE3) genome was accomplished and it resulted in a faster curcumin production in the initial 21 h, but after 63 h, the curcumin production by this mutant was 2.6 times lower as compared to the ‘original’ strain (i.e. the strain harboring the curcuminoids biosynthetic pathway but with no gene knockout). The same deletion in E. coli BL21 (DE3) genome resulted in a more significant decrease in curcumin production. In the future, the triple knock-out (ΔfumA,fumB,fumC) should be constructed to evaluate if curcumin production can indeed be improved as predicted in silico.
A curcumina tem sido reportada pelas suas propriedades benéficas incluindo como agente anticancerígeno. Apesar da curcumina apresentar um alto potencial terapêutico, tem uma baixa biodisponibilidade e é rapidamente metabolizada no organismo humano, o que implica uma repetitiva administração oral para atingir o efeito terapêutico pretendido. Além disso, a sua extração a partir das plantas é muito dispendiosa. Por estas razões, o uso de microrganismos para a produzir em larga escala e com melhores rendimentos constitui uma alternativa atraente. Neste sentido, Escherichia coli K-12 MG1655 (DE3) foi previamente geneticamente modificada adicionando três reações enzimáticas (4-cumarato-CoA ligase, dicetídeo-CoA sintase e curcumina sintase 1) que catalisam a produção de curcumina a partir do ácido ferúlico. No presente trabalho foi estabelecida a estirpe, as condições operacionais e a composição de meio ótimas para a produção de curcumina por E. coli contendo a via biossintética artificial. Anteriormente, utilizou-se uma estratégia comum de dois passos (LB+meio mínimo M9). Apesar de ser praticável numa escala laboratorial, a separação de biomassa é muito mais difícil, trabalhosa e dispendiosa numa fermentação em grande escala. Assim, foi implementada uma única formulação de meio mais adequada para a produção de curcumina a nível industrial. O meio mínimo MOPS, TB e LB foram avaliados. Usando as condições otimizadas, a concentração de curcumina produzida neste estudo foi mais elevada do que as previamente descritas na literatura, 686,7±59,7 µM em TB (43 h) e 822,6±28,1 µM em LB+M9 (63 h). Estes resultados foram obtidos usando a estirpe E. coli BL21 (DE3) que foi a identificada como melhor produtora após produzir 3,7 vezes mais curcumina do que a E. coli K-12 MG1655 (DE3). Além disso, a toxicidade da curcumina para células de E. coli foi avaliada. Os resultados mostraram que concentrações de curcumina acima de 400 µM influenciam negativamente o crescimento de E. coli. Adicionalmente, um dos objetivos do presente trabalho foi construir e validar vários mutantes de E. coli (p. ex., ΔfumA,fumB,fumC) que foram previamente identificados in silico como os mais promissores no sentido de aumentar a produção de curcumina a partir do ácido ferúlico. A deleção do gene fumB do genoma de E. coli K-12 MG1655 (DE3) foi efetuada e resultou numa produção mais rápida de curcumina nas 21 h iniciais, mas após 63 h, a produção de curcumina usando este mutante foi 2,6 vezes mais baixa do que a da estirpe que lhe deu origem. A mesma deleção no genoma de E. coli BL21 (DE3) resultou num decréscimo ainda mais significativo na produção de curcumina. No futuro, é necessário ainda construir o mutante triplo (ΔfumA,fumB,fumC) para avaliar se a produção de curcumina é efetivamente aumentada como previsto in silico.

Periodic drought is the primary limitation of plant growth and crop yield. The rise of water demand caused by the increase in world population and climate change, leads to one of the biggest challenges of modern agriculture: to increase food and feed production. De novo DNA methylation is a process regulated by small interfering RNA (siRNAs), which play a role in plant response and adaptation to abiotic stress. In the particular case of water deficit, growing evidences suggest a link between the siRNA pathways and drought response in the model legume Medicago truncatula. As a first step to understand the role of DNA methylation under water stress, we have set up several bioinformatics and molecular methodologies allowing the design of Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems and the assembly of TALENs (transcription activator-like effector nucleases), to target both dicer-like 3 (MtDCL3) and RNA-Dependent RNA polymerase (MtRDR2), enzymes of the RNA-directed DNA methylation pathway. TALENs efficiency was evaluated prior to plant transformation by a yeast-based assay using two different strategies to test TALENs activity: Polyacrylamide gel electrophoresis (PAGE) and Single strand conformation polymorphisms (SSCP). In this assay, yeast cells triple transformation emerged as good and rapid alternative to laborious yeast mating strategies. PAGE analysis might be a valuable tool to test TALENs efficacy in vivo if we could increase TALENs activity. SSCP-based approach proved to be ineffective due to the generation of several false positives. TALENs and CRISPR/Cas9 system constructed and designed in this work will in the future certainly enable the successful disruption of DCL3 and RDR2 genes and shed the light on the relationship between plant stress resistance and epigenetic regulation mediated by siRNAs in M.truncatula.