La leucémie myéloïde chronique (LMC) a été le premier cancer humain associé à une anomalie chromosomique : le chromosome de Philadelphie. Le gène de fusion BCR-ABL1 résultant code pour une tyrosine kinase ayant une activité dérégulée. Les inhibiteurs de tyrosine kinase (ITKs), qui inactivent la protéine BCR-ABL1, représentent la thérapie ciblée la plus efficace pour la LMC en phase chronique. Cependant, la LMC en phase avancée ne répond pas bien au traitement par les ITKs. Les mécanismes sous-jacents à la progression de la LMC ne sont pas bien compris. Par conséquent, la découverte de gènes qui coopèrent avec l’oncogène BCR-ABL1, et qui pourraient expliquer la progression de la LMC vers des phases avancées, est nécessaire pour l’identification de nouvelles cibles thérapeutiques de la LMC. Nous proposons d'établir un modèle cellulaire humain permettant l'identification de gènes capables de coopérer avec l'oncogène BCR-ABL1 pour une transformation tumorale complète. Ce système repose sur l'expression de BCR-ABL1 et l'inactivation d'autres gènes en particulier à l'aide de la technologie CRISPR-Cas9. L'identification des gènes coopérant avec BCR-ABL1 permettra la création de modèles cellulaires pour faciliter la sélection de médicaments capables de traiter la LMC dans les phases finales. Dans un second temps, une étude approfondie du gène TP53 a été menée. Ce gène étant muté dans plus de 50% des cancers, il est important de déterminer les conséquences de son inactivation dans des fibroblastes humains non tumoraux. La technologie CRISPR-Cas9 a été utilisée afin d’inactiver ce gène en particulier. Puis les cellules exprimant la forme sauvage ou la forme inactivée de p53 ont subi un traitement à la nutline-3a. Cette molécule empêche l’interaction du facteur de transcription p53 avec son inhibiteur MDM2. Des analyses transcriptomiques ont alors permis d’identifier d’une part les cibles aspécifiques de la nutline-3a et d’autre part les gènes cibles de p53 dans cette lignée de fibroblaste. / Chronic myeloid leukemia (CML) was the first human cancer to be consistently associated with a chromosomal abnormality: the Philadelphia chromosome. The resulting BCR-ABL1 fusion gene encodes a tyrosine kinase with deregulated activity. Tyrosine kinase inhibitors (TKIs) inactivating the BCR-ABL protein represent the most successful targeted therapy for CML in chronic phase. However, advanced CML does not respond well to TKIs treatment. The mechanisms underlying the progression of CML are not well understood. Therefore, the discovery of genes that cooperate with the BCR-ABL1 oncogene, which could explain the progression of CML to advanced phases, is required for the identification of novel therapeutic targets of CML. We propose to establish a human cellular model system that allows the identification of genes that are able to cooperate with the oncogene BCR-ABL1 for full tumoral transformation. This system relies on the expression of BCR-ABL1 and the generation of gene knock-out by using the CRISPR-Cas9 technology. Identification of genes cooperating with BCR-ABL1 will permit the creation of cellular model systems for identifying drugs that are able to treat CML in final phases. Secondly, we performed a detailed study of TP53 function. This gene is mutated in more than 50% of all cancer types. It is therefore crucial to understand the impact of its inactivation in human fibroblast cells. The CRISPR/Cas9 system was used to inactivate this gene. Wild-type and TP53 knock-out cells subsequently underwent nutlin-3a treatment. This molecule blocks the interaction between p53 and its regulator: MDM2. Transcriptomic analyses were performed to further study p53 regulated genes, and also to discover other potential nutlin-3a targets.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a major cause of Parkinson’s disease (PD), which is why modelling PD by replicating effects in animal models attracts great interest. However, the exact mechanisms of pathogenesis are still unclear. While a gain-of-function hypothesis generally receives consensus, there is evidence supporting an alternative loss-of-function explanation. Yet, neither overexpression of the human wild-type LRRK2 protein or its pathogenic variants, nor Lrrk2 knockout recapitulates key aspects of human PD in rodent models. Furthermore, there is conflicting evidence from morpholino knockdown studies in zebrafish regarding the extent of zygotic developmental abnormalities. Because reliable null mutants may be useful to infer gene function, and because the zebrafish is a more tractable laboratory vertebrate system than rodents to study disease mechanisms in vivo, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genomic editing was used to delete the ~60-kbp-long zebrafish lrrk2 locus containing the entire open reading frame. Constitutive removal of both the maternal and the zygotic lrrk2 function (mzLrrk2 individuals) causes a pleomorphic phenotype in the larval brain at 5 days post-fertilisation (dpf), including increased cell death, delayed myelination, and reduced and morphologically abnormal microglia/leukocytes. However, the phenotype is transient, spontaneously attenuating or resolving by 10 dpf, and the mutants are viable and fertile as adults. These observations are mirrored by whole-larva transcriptome data, revealing a more than eighteen-fold drop in the number of differentially expressed genes in mzLrrk2 larvae from 5 to 10 dpf. Additionally, analysis of spontaneous swimming activity shows hypokinesia as a predictor of Lrrk2 protein deficiency in larvae, but not in adult fish. Because the catecholaminergic (CA) neurons are the main clinically relevant target of PD in humans, the CA system of larvae and adult fish was analysed on both cellular and metabolic level. Despite an initial developmental delay at 5 dpf, the CA system is structurally intact at 10 dpf and later on in adult fish aged 6 and 11 months. However, monoamine oxidase (Mao)-dependent degradation of biogenic amines, including dopamine, is increased in older fish, possibly suggesting impaired synaptic transmission or a leading cause of cell damage in the long term. Furthermore, decreased mitosis rate in the larval brain was found, in the anterior portion only at 5 dpf, strongly and throughout the whole organ at 10 dpf. Conceivably, lrrk2 may have a more general role in the control of cell proliferation during early development and a more specialised one in the adult stage, possibly conditional, for example upon brain damage. Because the zebrafish can regenerate lost neurons, it represents a unique opportunity to elucidate the endogenous processes that may counteract neurodegeneration in a predisposing genetic background. To this aim, the regenerative potential of the adult telencephalon upon stab injury was tested in mzLrrk2 fish. Indeed, neuronal proliferation was reduced, suggesting that a complete understanding of Lrrk2 biology may not be fully appreciated without recreating challenging scenarios. To summarise, the present results demonstrate that loss of lrrk2 has an early effect on zebrafish brain development that is later often compensated. Nonetheless, perturbed aminergic catabolism, and specifically increased Mao-dependent aminergic degradation, is reported for the first time in a LRRK2 knockout model. Furthermore, a link between Lrrk2 and the control of basal cell proliferation in the brain, which may become critical under challenging circumstances such as brain injury, is proposed. Future directions should aim at exploring which brain cell types are specifically affected by the mzLrrk2 hypoproliferative phenotype and the resulting consequences on a circuitry level, particularly in very old fish (i.e., over 2 years of age).

La distribution des sucres est un processus clé dans le développement de la plante et cours des interactions plantes/microorganismes.Une recherche des acteurs moléculaires impliqués dans la répartition des ressources carbonées au cours de l'interaction avec le champignon nécrotrophe B. cinerea a été réalisée. Plusieurs familles de transporteurs de sucres et d'invertases ont été ciblées, permettant d'établir une cartographie des gènes régulés transcriptionnellement lors de l'interaction. Le rôle de certains gènes candidats a été étudié par une approche de génomique fonctionnelle afin de mettre en évidence une fonction biologique de l'allocation du carbone dans la résistance de la plante aux champignons nécrotrophes. Un système d'interaction simplifié, basé sur un dialogue moléculaire sans contact physique entre une culture cellulaire d'A. thaliana et B. cinerea, a été développé. Il a permis de mesurer les flux de sucres ainsi que les activités enzymatiques et métaboliques pour chaque partenaire. Nos résultats montrent que B. cinerea entraine une forte augmentation de l'activité invertasique pariétale dans les tissus infectés, indiquant qu'une transition source/puits a lieu. Plusieurs transporteurs de sucres sont différentiellement exprimés, certains d'entre eux modulant le devenir de l'interaction. L'activité d'absorption d'hexoses et le métabolisme primaire des cellules hôtes sont fortement stimulés, démontrant l'importance de la compétition pour les sucres à l'interface plante/agent pathogène. En conclusion, l'absorption des sucres alimente le métabolisme énergétique des cellules hôtes et participe aux mécanismes de défense de la plante. / During plant development and upon pathogen infection, sugar allocation is a key process in plant physiology. Cell wall invertases and sugar transporters, involved in the sink strength, likely play a major role in the metabolic plant response. Molecular actors involved in carbohydrates allocation upon B. cinerea interaction have been identified using a transcriptional approach. Some gene families of sugar transporters and invertases have been targeted, allowing the establishment of a cartography of genes regulated during the interaction. To understand the biological role of carbon allocation during the interaction between plants and necrotrophic fungi, candidate genes have been studied using a functional genomics approach.A simplified interaction system has been developped, allowing a molecular dialogue between Arabidopsis and B. cinerea cells, without any physical contact. This system enables the monitoring of radiolabelled sugar uptake rates and some enzymatic and metabolomic activities for both the host cells and the pathogen, independently.Globally, our results demonstrate that B. cinerea infection leads to the transition from a source to a sink tissue, with a strong increase in cell wall invertase activity. The expression of some sugar transporter genes is also affected, while some of them (AtSTP1 and 13) are involved in the disease development. Besides the increase in hexose uptake activity, primary metabolism is deeply affected, highlighting the competition for apoplastic sugars that takes place at the plant/pathogen interface. Sugar retrieval appears to be a key process, fuelling host cells with energy and signal molecules, contributing to the plant defense mechanisms.

Pseudomonas aeruginosa est un pathogène opportuniste responsable d’infections nosocomiales sévères associées à un taux élevé de mortalité. Le système de sécrétion de Type III (SST3) et les effecteurs qu’il injecte sont considérés comme des facteurs de virulence prépondérants de P. aeruginosa. Récemment nous avons caractérisé, un groupe de souches ne possédant pas les gènes du SST3, mais dont la virulence repose sur la sécrétion d’une nouvelle toxine de 172 kDa, nommée Exolysine (ExlA) qui provoque la perméabilisation de la membrane des cellules hôtes. ExlA est sécrétée dans le milieu par une porine de la membrane externe, nommée ExlB, formant ainsi un nouveau système de sécrétion à deux partenaires (TPS), ExlBA. Outre le domaine TPS du coté N-terminal de la protéine, impliqué dans sa sécrétion, ExlA possède différents domaines ; des répétitions hémagglutinines, cinq motifs Arginine-Glycine-Acide Aspartique (RGD) et un domaine C-Terminal faiblement conservé. Des tests de cytotoxicité sur des cellules eucaryotes ont montrés que la délétion du domaine C-terminal abolissait l’activité toxique d’ExlA. En utilisant un modèle de liposomes et différents types de cellules eucaryotes, comme les globules rouges, nous avons démontré qu’ExlA forme des pores membranaires de 1.6 nm. De plus, par un criblage cellulaire à haut-débit d’une banque de mutants obtenus par une mutagenèse de transposition, nous avons montré qu’un facteur bactérien additionnel était requis dans la toxicité d’ExlA. En effet, parmi les 7 400 mutants, nous avons identifiés 3 transposons insérés dans des gènes codant pour le pili de type IV, démontrant ainsi que cet appendice impliqué dans l’adhésion des bactéries participe à la toxicité d’ExlA, en permettant un contact rapproché entre la bactérie et les cellules hôtes. Un criblage de macrophages primaires de souris KO pour différentes protéines impliquées dans la voie de l’activation de l’inflammasome, nous a permis de démontrer que le pore formé par ExlA est responsable de l’activation de la Caspase-1 par l’inflammasome NLRP3 conduisant à la maturation de l’interleukine-1ß. Une étude bio-informatique a révélé la présence de gènes homologues à exlA chez d’autres espèces de Pseudomonas non pathogènes, comme P. putida, P. protegens, P. entomophila. Nous avons montré que ces bactéries environnementales sont aussi capables de provoquer une mort cellulaire dépendante de la Caspase-1. Finalement, un criblage d’une banque de macrophages dont les gènes ont été invalidés par la technologie CRISPR/cas9 a révélé que plusieurs protéines du système immunitaire, indirectement liées à l’activation de la Caspase-1 sont impliquées dans la mort cellulaire médiée par ExlA. De plus, nous avons montré que plusieurs sgRNAs ciblant un microARN, mir-741, était grandement enrichi dans les macrophages ayant résisté à une infection avec ExlA. Mir-741 régule l’expression d’enzymes (St8sIa1 et Agpat5) impliquées dans la voie de biosynthèse des sphingolipides et des glycérophospholipides, suggérant ainsi que l’activité d’ExlA requiert un environnement lipidique particulier. / Pseudomonas aeruginosa is a human opportunistic pathogen responsible for nosocomial infections associated with high mortality. The type III secretion system (T3SS) and T3SS-exported toxins have been considered as key infectivity virulence factors. Our team recently characterized a group of strains lacking T3SS, but employing a new pore-forming toxin of 172 kDa, named Exolysin (ExlA) that provokes cell membrane disruption. In this work we demonstrated that the ExlA secretion requires ExlB, a predicted outer membrane protein encoded in the same operon, showing that ExlA-ExlB define a new active Two-Partner Secretion (TPS) system. In addition to the TPS secretion signals, ExlA harbors several distinct domains, which comprise hemagglutinin domains, five Arginine-Glycine-Aspartic acid (RGD) motifs and a non-conserved C-terminal region lacking any identifiable sequence motifs. Cytotoxic assays showed that the deletion of the C-terminal region abolishes host-cell cytolysis. Using liposomes and eukaryotic cells, including red blood cells, we demonstrated that ExlA forms membrane pores of 1.6 nm. Based on a transposon mutagenesis strategy and a high throughput cellular live-dead screen, we identified additional bacterial factors required for ExlA-mediated cell lysis. Among 7 400 mutants, we identified three transposons inserted in genes encoding components of the Type IV pili, which are adhesive extracellular appendices. Type IV pili probably mediate close contact between bacteria and host cells and facilitate ExlA cytotoxic activity. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages to achieve host cell intoxication. Using mice primary bone marrow macrophages we showed that ExlA pores provoke activation of Caspase-1 via the NLRP3-inflamasomme followed by the maturation of the pro-interleukin-1ß. Mining of microbial genomic databases revealed the presence of exlA-like genes in other Pseudomonas species rarely associated with human infections P. putida, P. protegens and P. entomophila. Interestingly, we showed that these environmental bacteria are also able to provoke Caspase-1 cleavage and pro-inflammatory cell death of macrophages. Finally, genome-wide loss-of-function CRISPR/cas9 RAW library screen revealed that several components of the immune system response, indirectly linked to Caspase-1 are involved in the ExlA-mediated cell lysis. Moreover, we found at least three sgRNAs targeting miRNA, mir-741 were highly enriched in resistant macrophages challenged by ExlA. This miRNA regulates enzymes (St8sIa1 and Agpat5) in the sphingolipids and glycerophololipids biosynthesis pathways, suggesting that ExlA activity may require proper lipid environment.

Das plastidäre Genexpressionssystem höherer Pflanzen ist hochkomplex und differiert beträchtlich von dem prokaryotischer Vorfahren. Jeder einzelne Schritt der RNA-Prozessierung und Translation ist abhängig von nukleär kodierten, posttranslational in den Chloroplasten importierten Proteinen, insbesondere RNA-Bindeproteinen, wie den chloroplastidären Ribonukleoproteinen (cpRNP). Ein wichtiger und im Fokus dieser Arbeit stehender Vertreter ist CP29A, welcher ein breites Bindespektrum aufweist und in vivo mit einer Vielzahl von Transkripten interagiert. Frühere Studien belegen dennoch, dass ein Knockout von CP29A unter Standardkultivierungsbedingungen nicht in einem makroskopisch vom Wildtyp differierenden Phänotyp resultiert. Unter Kältestress hingegen ist CP29A für die Entwicklung photosynthetisch aktiver Chloroplasten essenziell. Zur tiefergehenden Charakterisierung der molekularen Funktion von CP29A wurde in der vorliegenden Arbeit eine genomweite Transkriptomanalyse der cp29a-Mutante durchgeführt. Es konnte erstmals gezeigt werden, dass CP29A bereits unter Standardkultivierungsbedingungen das Spleißen vieler plastidärer Introns unterstützt. Kälteinduziert weist das phänotypisch auffällige Gewebe der cp29a-Mutante eine globale Beeinträchtigung der Genexpression sowie massive Defekte der plastidären mRNA-Prozessierung auf. Da die Funktionalität von Proteinen substanziell von ihrer Lokalisation abhängig ist, wurden antikörperbasierte mikroskopische Lokalisationsstudien durchgeführt. Diese dokumentieren, dass CP29A kältestressinduziert zu dynamischen Granula lokalisiert, welche separiert von den plastidären Nukleoiden vorliegen und mit einem häufig in stressinduzierten Granula detektierten Protein kolokalisieren. / The plastid gene expression system in higher plants is highly complex and differs significantly from the gene expression system of the prokaryotic ancestors. Each individual step of RNA-processing and translation is dependent on nuclear-encoded RNA-binding proteins, such as chloroplast ribonucleoproteins (cpRNP), which are imported post-translationally into the chloroplast. An important representative is CP29A, which has a broad binding spectrum and interacts with a large number of transcripts in vivo. Earlier studies show that, while a knockout of CP29A under standard-cultivation-conditions does not result in a macroscopically different phenotype, under cold stress conditions CP29A is essential for the development of photosynthetically active chloroplasts. For a more detailed characterization of the molecular function of CP29A, a genome-wide transcriptome analysis of the cp29a mutant was carried out. It could be shown for the first time that CP29A already supports the splicing of many chloroplast introns under standard-cultivation-conditions. Cold-induced the phenotypically noticeable mutant tissue shows a global impairment of gene expression and massive defects in plastid mRNA processing. Since the functionality of proteins is substantially dependent on their localization, antibody-based microscopic localization studies were carried out. They reveal that cold stress-induced CP29A localizes to dynamic granules, which are separate from the plastid nucleoids and colocalize with a protein commonly detected in stress-induced granules.

Diabetes ist ein weltweites Problem, das durch den Verlust oder die Dysfunktion der Insulin-produzierenden β-Zellen des Pankreas verursacht wird. In seltenen Fällen entsteht Diabetes durch eine Mutation in einem einzigen Gen. Diese monogenetischen Formen des Diabetes können zur Identifizierung neuer Regulatoren der β-Zellen-Entwicklung und -Funktion beitragen. In der vorliegenden Arbeit habe ich neue putative Diabetes-assoziierte Gene untersucht, die zuvor durch „Next-Generation“ Sequenzierung in einer Gruppe von Kindern und Jugendlichen mit idiopathischem Diabetes festgestellt wurden. Insbesondere analysierte ich neuartige Mutationsvarianten in Genen kodierend für Histone deacetylase 4 (HDAC4), Glioma-associated oncogene homolog 1 (GLI1) und Glioma-associated oncogene homolog 2 (GLI2). Basierend auf den folgenden Kriterien wurden diese Transkriptionsregulatoren zur weiteren funktionellen Analyse priorisiert: Genetische Information, Patientenphänotyp und Expressionsprofil der Kandidaten Gene in Mauspankreas-Vorläuferzellen. Um die Rolle der Varianten während der pankreatischen Zelltypspezifizierung zu untersuchen, nutzte ich die CRISPR-Cas9 Methode in Kombination mit Stammzellendifferenzierung. Im Detail generierte ich diverse Stammzellen mittels CRISPR-Cas9, die die Mutationsvarianten der Patienten trugen und differenzierte diese zu β-ähnlichen Zellen. Weitere in vitro und Transkriptionsanalysen zeigten, dass die Variante c.C4661T in GLI2 die Entwicklung der β-ähnlichen Zellen beeinträchtigte, was für eine genetische Prädisposition zur Entwicklung von Diabetes verantwortlich sein kann. Zusätzlich nutzte ich diese Plattform, um neue extrinsische Faktoren zu untersuchen und zeigte, dass die fördernde Rolle von HC toxin (HDAC Inhibitor) und SLIT3 (ROBO Ligand) konserviert ist. Zusammenfassend habe ich eine Differenzierungsplattform etabliert, um die Rolle von genetischen und extrinsischen Faktoren für die Entwicklung des Pankreas und/oder β-Zellen zu untersuchen. / Diabetes is a worldwide health problem caused by the loss or dysfunction of the insulin-secreting β-cells in the pancreas. Unelucidated forms of monogenic diabetes, arising from rare mutations in one single gene, represent invaluable models for identifying new targets of β-cell development and function. In this study, I focused on putative disease-associated genes for diabetes that have been previously identified by next-generation sequencing of a cohort of patients with puberty-onset diabetes. In particular, I investigated unique mutant variants in genes coding for Histone deacetylase 4 (HDAC4), Glioma-associated oncogene homolog 1 (GLI1) and Glioma-associated oncogene homolog 2 (GLI2). These transcriptional regulators were prioritized for functional analysis based on patient phenotype, expression level in pancreas progenitor cells and available genetic information. To investigate the role of the genetic mutant variants in pancreatic cell fate decisions and cell function, I used the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 genome editing technology in combination with human induced pluripotent stem cell (iPSC)-directed β-cell differentiation. Employing these approaches, I established several patient-like iPSC lines carrying the identified heterozygous missense variants. Specifically, functional experiments and whole transcriptome analysis showed that the variant c.C4661T in GLI2 impairs human β-cell differentiation and β-cell function, which might be responsible for a genetic predisposition to develop diabetes. In addition, I used the same iPSC-based differentiation model system to study novel extrinsic factors, namely the HDAC inhibitor HC toxin and the ROBO ligand SLIT3 and uncovered their conserved role in enhancing human β-cell development. Taking together, I established a human iPSC differentiation platform to study critical genes and extrinsic factors that are necessary for human pancreas development and/or β-cells.

Wie regulatorische Sequenzen die Genexpression steuern, ist von grundlegender Bedeutung für die Erklärung von Phänotypen in Gesundheit und Krankheit. Die Funktion regulatorischer Sequenzen muss letztlich in ihrer genomischen Umgebung und in entwicklungs- oder gewebespezifischen Zusammenhängen verstanden werden. Da dies eine technische Herausforderung ist, wurden bisher nur wenige regulatorische Elemente in vivo charakterisiert. Hier verwenden wir Induktion von Cas9 und multiplexed-sgRNAs, um hunderte von Mutationen in Enhancern/Promotoren und 3′ UTRs von 16 Genen in C. elegans zu erzeugen. Wir quantifizieren die Auswirkungen von Mutationen auf Genexpression und Physiologie durch gezielte RNA- und DNA-Sequenzierung. Bei der Anwendung unseres Ansatzes auf den 3′ UTR von lin-41, bei der wir hunderte von Mutanten erzeugen, stellen wir fest, dass die beiden benachbarten Bindungsstellen für die miRNA let-7 die lin-41-Expression größtenteils unabhängig voneinander regulieren können, mit Hinweisen auf eine mögliche kompensatorische Interaktion. Schließlich verbinden wir regulatorische Genotypen mit phänotypischen Merkmalen für mehrere Gene. Unser Ansatz ermöglicht die parallele Analyse von genregulatorischen Sequenzen direkt in Tieren. / How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. The function of regulatory sequences must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3′ UTRs of 16 genes in C. elegans. We quantify the impact of mutations on expression and physiology by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3′ UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression largely independently of each other, with indications of a compensatory interaction. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of gene regulatory sequences directly in animals.

Background: Type I interferonopathies comprise a heterogenous and phenotypically diverse range of diseases, characterized by an elevated level of type I interferon (IFN) exhibited in patients accompanied by high interferon stimulated gene (ISG) scores. Type I interferonopathies are difficult to treat, especially in the acute phases of the disease, and typically chronic, requiring lifelong treatment and care. A patient, exhibiting symptoms of a type I interferonopathy was identified by whole exome sequencing to have a compound heterozygous mutation in the type III IFN receptor, IFNLR1. The compound mutation is comprised of two discrete truncating mutations, c.532_535dupCATG (p.G179AfsX37) and c.904dupG (p.V302GfsX30), termed mutant 1 or mutant 2 (M1, M2), respectively. The M1 and M2 IFNLR1 proteins were shown to be expressed, but were demonstrated to be non-functional. Hypothesis: Mutant isoforms of IFNLR1 interfere with the normal function of the closely related IL-10 family of cytokines and receptors through a shared β subunit receptor chain IL10RB. It is hypothesized that M1 and M2 IFNLR1 are able to impair correct IL-10 or IL-22 receptor formation by preventing coupling with IL10RB resulting in immune dysregulation. Materials and Methods: Multiple CRISPR-Cas9 tools were implemented to create several cell lines with genome edits up to a single base pair resolution to model deficiencies in each of the IFN receptors: IFNLR1, IFNGR1, and IFNAR1. The knock-out cell lines were used as models for the expression of M1 and M2 IFNLR1, IL10RA, and IL10RB, to study the relationship between the IFN-λ, IL-10 and IL-22 pathways. Stimulation of these model pathways and expression systems with IL-10, IL-22, and IFN-λ helped further our understanding between these proteins. The relationship between the IL-10 and IFN-λ pathways was further explored by stimulation of whole blood derived from the patient, parents, and controls which was conducted to further quantify the role IL-10 signalling played in the pathogenesis of the disease. Results: M1 was demonstrated to promote the spontaneous phosphorylation of STAT1, STAT2, and STAT3 independent of stimulation. This phosphorylation was independent of type I, type II, or type III IFN signalling, with phosphorylation persisting in knock-out lines of all IFN receptors singly or in combination. Consequently, the closely related IL-10 family of cytokines was examined for its role in the pathogenesis of the disease. M1 and M2 were demonstrated to interact with IL10RA and IL10RB on the protein level and were demonstrated to influence the phosphorylation of STAT3 by IL-10 or IL-22 stimulation. Further analysis of whole blood derived from the patient, parents and controls demonstrated a lack of IL-10-mediated regulation of IL-12 solely in the patient. Elevated basal and stimulatory levels of IL-18, CXCL10, and IFN-γ were also detected in the patient. Conclusions: The patient maintained IL-10 regulatory capacity in all but IL-12 signalling, which is a pathway known to be directly controlled by IL-10. IL-12 is mainly produced in cells like dendritic cells, which are one of the only cell types to naturally express IFNLR1, IL10RA and IL10RB. The loss of IL-12 regulation by IL-10 likely stems from interference by the M1 and M2 IFNLR1 present in patient dendritic cells, inhibiting proper formation of the IL-10 receptor and preventing its regulatory function. The elevated levels of IL-12 in conjunction with elevated IL-18 levels, which functions synergistically with IL-12 result in secretion of high levels of IFN-γ. IFN-γ likely participates in a positive feedback loop with CXCL10, resulting in prolonged and heightened immune response after immune challenge in the patient resulting in autoinflammation.:List of Tables v List of Figures vi List of Abbreviations ix 1 Introduction 1 1.1 CRISPR-Cas9-Mediated Editing 1 1.1.1 Guide Efficiency and Off-Target Prediction 11 1.2 Type I Interferonopathies 13 1.3 Pathogen Detection by the Innate Immune System 14 1.3.1 TLR Dependent Nucleic Acid Sensing 14 1.3.2 Non-TLR-Mediated Detection of Nucleic Acids 17 1.4 Interferon-Mediated Innate Immunity 19 2 Hypotheses and Goals 24 2.1 Hypotheses 24 2.2 Goals 24 3 Methods and Materials 26 3.1 Table of Materials and Software Used 26 3.2 Cell Culture 30 3.2.1 Adherent Cell Culture 31 3.2.2 Suspension Cell Culture 31 3.2.3 Cell Counting and Seeding 32 3.2.4 Cell Defrosting and Freezing 33 3.2.5 Cytokine Stimulation 34 3.2.6 Transfection 35 3.3 Target Prediction 37 3.4 CRISPR-Cas9 Cloning 39 3.4.1 Insert Generation, Plasmid Digestion and Ligation 40 3.4.2 Transformation and Clonal Selection 43 3.4.3 In-Fusion Cloning 44 3.4.4 DNA-Miniprep 44 3.4.5 DNA-Maxiprep 45 3.5 PCR 46 3.5.1 DNA Extraction 47 3.5.2 Amplification Reaction (Standard PCR) 48 3.5.3 PCR Clean 49 3.5.4 In Vitro sgRNA Synthesis 50 3.5.5 pegRNA Templates 52 3.6 Measurement of DNA and RNA Concentration 54 3.7 Agarose Gel Electrophoresis 54 3.7.1 Gel Preparation 54 3.7.2 Electrophoresis Parameters 55 3.7.3 Gel Extraction 55 3.8 Gene Editing 57 3.8.1 CRISPR-Cas9-Mediated Gene Editing 57 3.8.2 Cutting Assays 60 3.8.3 Isolation of Single Clones 62 3.9 Sanger Sequencing 64 3.10 Immunostaining 64 3.10.1 Protein Extraction and BCA Assay 64 3.10.2 Western Blotting 66 3.10.3 Co-Immunoprecipitation 69 3.11 Whole Blood Assays 71 3.11.1 Whole Blood Stimulation 71 3.11.2 Flow Cytometry 72 3.11.3 Statistical Analysis 73 4 Results 74 4.1 Implementation of CRISPR-Cas9 Techniques 74 4.2 In Vitro sgRNA Synthesis for CRISPR-Cas9 Editing 75 4.3 Validation of CRISPR-Cas9 Editing 75 4.4 Cas9 Editing 78 4.4.1 IFNLR1 Editing 78 4.4.2 IFNLR1 Rescue 80 4.4.3 IFNGR1 Knock-Outs 81 4.4.4 IFNAR1 Knock-Outs 82 4.4.5 Targeted Installation of Mutations 84 4.5 Investigation of a Type I Interferonopathy 88 4.5.1 Characterization of a Patient with Complete IFNLR1 Deficiency 88 4.5.2 Basal Level of pSTAT1 and pSTAT3 in Patient Cells 89 4.5.3 IFNLR1 Expression in Patient 91 4.5.4 Overexpression of IFNLR1 Isoforms 93 4.5.5 Spontaneous Induction of STAT1 Phosphorylation 94 4.5.6 Independence of Immune Activation from IFN Signalling Pathways 96 4.6 IL-10 and IL-22 Stimulations 98 4.6.1 IL-10 Stimulation 98 4.6.2 Interaction of IL10RA, IL10RB, and IFNLR1 Isoforms 102 4.6.3 IL-22 Stimulation 106 4.7 Whole Blood Assays 107 4.7.1 TNF-α 107 4.7.2 IFN-γ 108 4.7.3 IL-12 and IL-18 109 4.7.4 CXCL10 111 4.7.5 IL-10 112 5 Discussion 114 5.1 Cell Model Creation by CRISPR-Cas9 Techniques 114 5.1.1 Cutting Assays and CRISPR-Cas9 Validation 114 5.1.2 IFN Receptor Knock-outs 116 5.1.3 Base Editing and Prime Editing 117 5.2 Characterization of a Complete IFN-λ Receptor Deficiency 121 5.2.1 Establishing an IFNLR1 Overexpression System 121 5.2.2 IL-10 Family of Cytokines 124 5.3 Whole Blood Assays 129 6 Conclusions 133 7 Summary 135 8 Zusammenfassung 137 9 Scientific Output 139 10 Literature 140 11 Acknowledgements 155 12 Appendix 156 12.1.1 Supplementary Tables 156 12.1.2 Supplementary Figures 160 12.1.3 Declarations 161 / Hintergrund: Typ-I-Interferonopathien umfassen ein heterogenes und phänotypisch vielfältiges Spektrum von Krankheiten, die sich durch einen erhöhten Typ-I-Interferon (IFN)-Spiegel bei Patienten auszeichnen, der mit einer hohen Expression IFN-stimulierter Gene (ISG) einhergeht. Typ-I-Interferonopathien sind vor allem in den akuten Phasen der Krankheit oft schwer zu behandeln und verlaufen in der Regel chronisch, so dass eine lebenslange Behandlung erforderlich ist. Bei einer Patientin mit Symptomen einer Typ-I-Interferonopathie wurde durch eine Exom-Sequenzierung eine compound heterozygote Mutation im IFNLR1-Gen, das den Typ-III-IFN-Rezeptor bzw. INF-λ-Rezeptor kodiert, festgestellt. Dabei handelt es sich um zwei trunkierende Mutationen, c.532_535dupCATG (p.G179AfsX37) und c.904dupG (p.V302GfsX30), die als Mutante 1, beziehungsweise Mutante 2 (M1, M2) bezeichnet wurden. Es konnte gezeigt werden, dass die M1- und M2-IFNLR1-Proteine zwar exprimiert wurden, allerdings nicht funktionell waren. Hypothese: Mutierte Isoformen von IFNLR1 beeinträchtigen die normale Funktion der eng verwandten Interleukin-10-Familie von Zytokinen und Rezeptoren durch die gemeinsame β Untereinheit, der Rezeptorkette IL10RB. Es wird angenommen, dass M1 und M2 die korrekte IL-10 oder IL-22-Rezeptorbildung beeinträchtigen, indem sie die Kopplung mit IL10RB verhindern, was zu einer Dysregulation des Immunsystems führt. Material und Methode: Mittels verschiedener CRISPR-Cas9-Methoden, teilweise mit einer Genauigkeit bis zu einem Basenpaar, wurden mehrere Zelllinien mit editierten Gensequenzen erzeugt, um Funktionsverluste der verschiedenen IFN-Rezeptoren, IFNLR1, IFNGR1 und IFNAR1, zu modellieren. Die Knock-out-Zelllinien wurden dann als Modelle für die Expression von M1 und M2 IFNLR1 sowie IL10RA und IL10RB verwendet, um die Beziehung zwischen den IFN-λ, IL-10 und IL 22-Signalwegen zu untersuchen. Die Untersuchung dieser modellierten Expressionssysteme mit IL-10, IL 22 und IFN-λ trug zu einem besseren Verständnis der Beziehungen zwischen diesen Proteinen bei. Die Beziehung zwischen den IL-10 und IFN λ Signalwegen wurde durch die Stimulierung von Blutproben der Patientin, deren Eltern, sowie von Kontrollpersonen weiter untersucht, um die Rolle der IL-10-Signalübertragung bei der Pathogenese der Autoinflammation weiter zu chrakterisieren. Ergebnisse: Es wurde gezeigt, dass M1 zu einer spontanen, stimulationsunabhängigen Phosphorylierung von STAT1, STAT2 und STAT3 führt. Diese Phosphorylierung zeigte sich unabhängig vom Typ-I-, Typ-II- oder Typ III IFN Signalweg, wobei die Phosphorylierung in Knock-out-Zelllinien aller IFN Rezeptoren, einzeln oder in Kombination, bestehen blieb. Daraufhin wurde die eng verwandte IL-10-Familie von Zytokinen auf ihre Rolle bei der Pathogenese der Krankheit untersucht. Es wurde nachgewiesen, dass M1 und M2 mit IL10RA und IL10RB auf Proteinebene interagieren und die Phosphorylierung von STAT3 durch IL-10 oder IL-22 Stimulation beeinflussen. Weitere Analysen des Vollbluts der Patientin, der Eltern und von Kontrollpersonen zeigten, dass IL-10 die Regulation von IL-12 einzig bei der Patientin nicht beeinflusste. Ebenfalls wurden bei der Patientin erhöhte Basal- und Stimulationswerte von IL 18, CXCL10 und IFN-γ festgestellt. Schlussfolgerungen: Die Patientin behielt die regulierende Funktion von IL-10 in allen Bereichen bei, mit Ausnahme des IL-12-Signalwegs, von dem bekannt ist, dass er direkt durch IL-10 kontrolliert wird. IL-12 wird hauptsächlich in dendritischen Zellen produziert, die zu den einzigen Zelltypen gehören, die natürlicherweise IFNLR1, IL10RA und IL10RB exprimieren. Der Verlust der IL 12 Regulierung durch IL-10 ist wahrscheinlich auf eine Störung durch die in den dendritischen Zellen der Patientin vorhandenen M1- und M2-IFNLR1-Mutationen zurückzuführen, die die ordnungsgemäße Bildung des IL-10-Rezeptors hemmen und seine Regulierungsfunktion verhindern. Die erhöhten IL-12-Spiegel in Verbindung mit erhöhten IL-18-Spiegeln, die synergistisch mit IL-12 wirken, führen zu einer hohen Sekretion von IFN-γ. ermutlich ist IFN γ an einer positiven Rückkopplungsschleife mit CXCL10 beteiligt, die nach einer Stimulierung des Immunsystems zu einer verlängerten und verstärkten Immunantwort bei der Patientin führt, die wiederum in einer Autoinflammation resultiert.:List of Tables v List of Figures vi List of Abbreviations ix 1 Introduction 1 1.1 CRISPR-Cas9-Mediated Editing 1 1.1.1 Guide Efficiency and Off-Target Prediction 11 1.2 Type I Interferonopathies 13 1.3 Pathogen Detection by the Innate Immune System 14 1.3.1 TLR Dependent Nucleic Acid Sensing 14 1.3.2 Non-TLR-Mediated Detection of Nucleic Acids 17 1.4 Interferon-Mediated Innate Immunity 19 2 Hypotheses and Goals 24 2.1 Hypotheses 24 2.2 Goals 24 3 Methods and Materials 26 3.1 Table of Materials and Software Used 26 3.2 Cell Culture 30 3.2.1 Adherent Cell Culture 31 3.2.2 Suspension Cell Culture 31 3.2.3 Cell Counting and Seeding 32 3.2.4 Cell Defrosting and Freezing 33 3.2.5 Cytokine Stimulation 34 3.2.6 Transfection 35 3.3 Target Prediction 37 3.4 CRISPR-Cas9 Cloning 39 3.4.1 Insert Generation, Plasmid Digestion and Ligation 40 3.4.2 Transformation and Clonal Selection 43 3.4.3 In-Fusion Cloning 44 3.4.4 DNA-Miniprep 44 3.4.5 DNA-Maxiprep 45 3.5 PCR 46 3.5.1 DNA Extraction 47 3.5.2 Amplification Reaction (Standard PCR) 48 3.5.3 PCR Clean 49 3.5.4 In Vitro sgRNA Synthesis 50 3.5.5 pegRNA Templates 52 3.6 Measurement of DNA and RNA Concentration 54 3.7 Agarose Gel Electrophoresis 54 3.7.1 Gel Preparation 54 3.7.2 Electrophoresis Parameters 55 3.7.3 Gel Extraction 55 3.8 Gene Editing 57 3.8.1 CRISPR-Cas9-Mediated Gene Editing 57 3.8.2 Cutting Assays 60 3.8.3 Isolation of Single Clones 62 3.9 Sanger Sequencing 64 3.10 Immunostaining 64 3.10.1 Protein Extraction and BCA Assay 64 3.10.2 Western Blotting 66 3.10.3 Co-Immunoprecipitation 69 3.11 Whole Blood Assays 71 3.11.1 Whole Blood Stimulation 71 3.11.2 Flow Cytometry 72 3.11.3 Statistical Analysis 73 4 Results 74 4.1 Implementation of CRISPR-Cas9 Techniques 74 4.2 In Vitro sgRNA Synthesis for CRISPR-Cas9 Editing 75 4.3 Validation of CRISPR-Cas9 Editing 75 4.4 Cas9 Editing 78 4.4.1 IFNLR1 Editing 78 4.4.2 IFNLR1 Rescue 80 4.4.3 IFNGR1 Knock-Outs 81 4.4.4 IFNAR1 Knock-Outs 82 4.4.5 Targeted Installation of Mutations 84 4.5 Investigation of a Type I Interferonopathy 88 4.5.1 Characterization of a Patient with Complete IFNLR1 Deficiency 88 4.5.2 Basal Level of pSTAT1 and pSTAT3 in Patient Cells 89 4.5.3 IFNLR1 Expression in Patient 91 4.5.4 Overexpression of IFNLR1 Isoforms 93 4.5.5 Spontaneous Induction of STAT1 Phosphorylation 94 4.5.6 Independence of Immune Activation from IFN Signalling Pathways 96 4.6 IL-10 and IL-22 Stimulations 98 4.6.1 IL-10 Stimulation 98 4.6.2 Interaction of IL10RA, IL10RB, and IFNLR1 Isoforms 102 4.6.3 IL-22 Stimulation 106 4.7 Whole Blood Assays 107 4.7.1 TNF-α 107 4.7.2 IFN-γ 108 4.7.3 IL-12 and IL-18 109 4.7.4 CXCL10 111 4.7.5 IL-10 112 5 Discussion 114 5.1 Cell Model Creation by CRISPR-Cas9 Techniques 114 5.1.1 Cutting Assays and CRISPR-Cas9 Validation 114 5.1.2 IFN Receptor Knock-outs 116 5.1.3 Base Editing and Prime Editing 117 5.2 Characterization of a Complete IFN-λ Receptor Deficiency 121 5.2.1 Establishing an IFNLR1 Overexpression System 121 5.2.2 IL-10 Family of Cytokines 124 5.3 Whole Blood Assays 129 6 Conclusions 133 7 Summary 135 8 Zusammenfassung 137 9 Scientific Output 139 10 Literature 140 11 Acknowledgements 155 12 Appendix 156 12.1.1 Supplementary Tables 156 12.1.2 Supplementary Figures 160 12.1.3 Declarations 161

Development and regeneration of the vertebrate eye are the result of complex interactions of regulatory networks and spatiotemporally controlled gene expression events. During embryonic retinal development, the coordination of cell signaling and transcriptional regulation allows for a relatively homogenous sheet of neuroepithelial cells to proliferate and differentiate in-to a multilayered, light sensitive retinal tissue. Following injury, the retinas of many cold-blooded vertebrates, such as the zebrafish, undergo a proliferative response that results not only in new retinal cells of the correct type in the correct location, but also functional integration of these cells and restoration of vision. In order for embryonic retinal neurogenesis to proceed correctly, systems must be in place that restrict subsets of progenitor cells from differentiation. Pools of actively proliferating retinal progenitor cells are maintained to fill the needs of developmental processes and normal growth of the retina. In addition, subsets of radial glia in the retina retain the ability to de-differentiate into proliferating progenitor cells to meet the demands of the regenerating retina. All of these processes rely on the tight coordination of extrinsic and intrinsic cues, as well as regulation of gene expression by transcription factors. Although a considerable amount of work has been conducted to identify key regulators of retinal development and regeneration, many gene regulatory networks which include both master signaling pathways as well as individual transcription factors remain poorly characterized. Some of these factors implicated in retinal development and regeneration are members of the Hairy/Enhancer of Split (Hes) superfamily of genes, including the Hairy-related (Her) factors Her4 and Her9. Her transcription factors are basic-helix-loop-helix-orange (bHLH-O) transcription factors that bind to palindromic E- and N-box canonical sequences in the promoters of target genes. Her factors have been previously shown to play roles in a diverse array of developmental and neurogenic processes, including neural tube closure, floor plate development, somitogenesis, and development of various components of the central nervous system as well as the cranial sensory placodes. The roles of her4 and her9 in retinogenesis, however, remain undefined. To determine the possible roles of her4 and her9 factors in the retina, I characterized the expression patterns of these factors during developmental retinal neurogenesis and/or regeneration, examined loss of function phenotypes, and identified signaling pathways that modulate expression of these factors. Chapter 1 of this dissertation provides an overview of vertebrate retina and retinal development, the known functions of her4 in other tissues, and the Notch-Delta signaling pathway. Chapter 2 provides evidence that her4 is a primary effector of the Notch pathway during retinal development, and examines the role of her4 expressing cells during regeneration of the mature zebrafish retina within the context of both chronic and acute photoreceptor damage paradigms. In addition, generation and validation of the transgenic her4:Kaede zebrafish which was used to identify the lineage of her4-expressing cells is described. Characterization of her9 during retinal development, identification of the retinoic acid signaling pathway as a regulator of her9 expression in the retina, and the role her9 plays during retinal vasculogenesis are discussed in Chapter 3. Chapter 4 discusses the generation of her9 knock-out zebrafish lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and characterization of mutant phenotypes in mosaic her9 mutant F0 fish. In addition, in Chapter 4 I also discuss the screening processes used to identify and characterize genetic lesions in the her9 allele and establish various lines that stably transmit deleterious her9 alleles in the germline, and provide preliminary data of the her9 mutant phenotype. Finally, in Chapter 5 I discuss conclusions from the data generated from this dissertation, additional studies that would expand upon this work, and the implications of these results on the broader understanding of retinal development and regeneration. My dissertation incorporates reverse genetic analysis in zebrafish, biochemical analysis, transgenesis, and various molecular approaches to help better understand the roles of her4 and her9 during retinal neurogenesis. Moreover, these studies may also contribute to a better understanding of retinal development, and disease pathogenesis. It is my hope that this work could also ultimately contribute, even if in some small way, to the goal of enabling human patients who have suffered from vision loss a means by which a damaged retina could be regenerated and functional vision restored.

<p>Duchenne muscular dystrophy (DMD) is a severe hereditary disorder caused by a loss of dystrophin, an essential musculoskeletal protein. Decades of promising research have yielded only modest gains in survival and quality of life for these patients and there have been no approved gene therapies for DMD to date. There are two significant hurdles to creating effective gene therapies for DMD; it is difficult to deliver a replacement dystrophin gene due to its large size and current strategies to restore the native dystrophin gene likely require life-long administration of a gene-modifying drug. This thesis presents a novel method to address these challenges through restoring dystrophin expression by genetically correcting the native dystrophin gene using engineered nucleases that target one or more exons in a mutational hotspot in exons 45-55 of the dystrophin gene. Importantly, this hotspot mutational region collectively represents approximately 62% of all DMD mutations. In this work, we utilize various engineered nuclease platforms to create genetic modifications that can correct a variety of DMD patient mutations.</p><p>Initially, we demonstrate that genome editing can efficiently correct the dystrophin reading frame and restore protein expression by introducing micro-frameshifts in exon 51, which is adjacent to a hotspot mutational region in the dystrophin gene. Transcription activator-like effector nucleases (TALENs) were engineered to mediate highly efficient gene editing after introducing a single TALEN pair targeted to exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from DMD patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. We show that our engineered TALENs have minimal cytotoxicity and exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein coding regions of the genome, as predicted by in silico target site analysis. </p><p>In an alternative approach, we capitalized on the recent advances in genome editing to generate permanent exclusion of exons by using zinc-finger nucleases (ZFNs) to selectively remove sequences important in specific exon recognition. This strategy has the advantage of creating predictable frame restoration and protein expression, although it relies on simultaneous nuclease activity to generate genomic deletions. ZFNs were designed to remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript, a method that can potentially restore the dystrophin reading frame in up to 13% of DMD patients. Nucleases were assembled by extended modular assembly and context-dependent assembly methods and screened for activity in human cells. Selected ZFNs had moderate observable cytotoxicity and one ZFN showed off-target activity at two chromosomal loci. Two active ZFN pairs flanking the exon 51 splice acceptor site were transfected into DMD patient cells and a clonal population was isolated with this region deleted from the genome. Deletion of the genomic sequence containing the splice acceptor resulted in the loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin expression in vitro. Furthermore, transplantation of corrected cells into the hind limb of immunodeficient mice resulted in efficient human dystrophin expression localized to the sarcolemma. </p><p>Finally, we exploited the increased versatility, efficiency, and multiplexing capabilities of the CRISPR/Cas9 system to enable a variety of otherwise challenging gene correction strategies for DMD. Single or multiplexed sgRNAs were designed to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45-55 and introducing either intraexonic small insertions and deletions, or large deletions of one or more exons. Significantly, we generated a large deletion of 336 kb across the entire exon 45-55 region that is applicable to correction of approximately 62% of DMD patient mutations. We show that, for selected sgRNAs, CRISPR/Cas9 gene editing displays minimal cytotoxicity and limited aberrant mutagenesis at off-target chromosomal loci. Following treatment with Cas9 nuclease and one or more sgRNAs, dystrophin expression was restored in Duchenne patient muscle cells in vitro. Human dystrophin was detected in vivo following transplantation of genetically corrected patient cells into immunodeficient mice. </p><p>In summary, the objective of this work was to develop methods to genetically correct the native dystrophin as a potential therapy for DMD. These studies integrate the rapid advances in gene editing technologies to create targeted frameshifts that restore the dystrophin gene around patient mutations in non-essential coding regions. Collectively, this thesis presents several gene editing methods that can correct patient mutations by modification of specific exons or by deletion of one or more exons that results in restoration of the dystrophin reading frame. Importantly, the gene correction methods described here are compatible with leading cell-based therapies and in vivo gene delivery strategies for DMD, providing an avenue towards a cure for this devastating disease.</p> / Dissertation