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Bernheim, Aude. "The distribution of CRISPR-Cas systems is affected by interactions with DNA repair pathways." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB070/document.
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Les systèmes CRISPR-Cas confèrent aux bactéries une immunité adaptative contre les éléments génétiques mobiles jouant ainsi un rôle important dans l’évolution bactérienne. Cependant, moins de la moitié des génomes bactériens encodent des systèmes CRISPR-Cas ; cela, malgré la protection qu’ils confèrent et leur haut taux de transfert horizontal. Des hypothèses telles que le coût des phénomènes d’auto-immunité ou de posséder des défenses adaptatives plutôt qu’innées ont été mises en avant pour expliquer ce paradoxe. Je propose une nouvelle hypothèse complémentaire : le contexte génétique jouerait un rôle important dans la fixation d’un système CRISPR-Cas après son transfert. Plus précisément, j’ai étudié comment les interactions entre les systèmes de réparation de l’ADN et les CRISPR-Cas influencent la distribution de ces derniers. Pour cela, j’ai d’abord examiné finement la distribution des systèmes CRISPR-Cas dans les génomes bactériens. J’ai ensuite analysé les co-occurences des systèmes de réparation de l’ADN et des CRISPR-Cas et démontré l’existence d’associations positives et négatives entre eux. Enfin, je me suis concentrée sur une des associations négatives découvertes pour valider mes prédictions expérimentalement et comprendre les mécanismes moléculaires sous-jacents. Mes travaux permettent de mieux comprendre les interactions complexes entre systèmes de réparation de l’ADN et CRISPR-Cas et démontrent la nécessite d’accommodation des CRISPR-Cas à un contexte génétique pour être sélectionnés et maintenus dans les génomes bactériensCRISPR-Cas systems confer bacteria and archea an adaptative immunity against phages and other invading genetic elements playing an important role in bacterial evolution. Only 47% of bacterial genomes harbor a CRISPR-Cas system despite their high rate of horizontal transfer. Hypothesis such as the cost of autoimmu- nity or the trade off between a constitutive or an inducible defense system have been put forward to explain this paradox. I propose that the genetic background plays an important role in the process of maintaining a CRISPR-Cas system af- ter its transfer. More precisely I hypothesized that CRISPR-Cas systems interact with DNA repair pathways. To test this idea, we detected DNA repair pathways and CRISPR-Cas systems in bacterial genomes and studied their co-occurences. We report both positive and negative associations that we interpret as poten- tial antagonistic or synergistic interactions. We then focused on one interaction to validate our result experimentally and explored molecular mechanisms behind those interactions. My findings give insights on the complex interactions between CRISPR-Cas systems and DNA repair mechanisms in bacteria and provide a first example on the necessity of accommodation of CRISPR-Cas systems to a specific genetic context to be selected and maintained in bacterial genomes
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Vyhovskyi, Danylo. "In vivo studies of CRISPR adaptation mechanism and specificity." Electronic Thesis or Diss., Sorbonne université, 2023. http://www.theses.fr/2023SORUS729.
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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 efficacesThis 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
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Alkhnbashi, Omer S. [Verfasser], and Rolf [Akademischer Betreuer] Backofen. "Computational characterisation of genomic CRISPR-Cas systems in archaea and bacteria." Freiburg : Universität, 2017. http://d-nb.info/1139210904/34.
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Ansai, Satoshi. "Targeted mutagenesis in medaka using targetable nuclease systems." Kyoto University, 2016. http://hdl.handle.net/2433/215591.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第19765号
農博第2161号
新制||農||1039(附属図書館)
学位論文||H28||N4981(農学部図書室)
32801
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 田川 正朋
学位規則第4条第1項該当
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Zhu, Houxiang. "Optimal gRNA design of different CRISPR-Cas systems for DNA and RNA editing." Miami University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=miami1556307865151938.
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Brendel, Jutta [Verfasser]. "Charakterisierung der Prozessierungs-und Interferenzaktivität des CRISPR/Cas-Systems in Haloferax volcanii / Jutta Brendel." Ulm : Universität Ulm. Fakultät für Naturwissenschaften, 2014. http://d-nb.info/1053705611/34.
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Hille, Frank [Verfasser]. "Investigation of Spacer Acquisition Mechanisms in Type V-A CRISPR-Cas Systems / Frank Hille." Berlin : Humboldt-Universität zu Berlin, 2020. http://d-nb.info/1215570341/34.
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Staub, Dillon. "Bio-Inspired Hardware Security Defenses: A CRISPR-Cas-Based Approach for Detecting Trojans in FPGA Systems." University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1563872470616901.
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Silva, Caroline Caetano da. "O nr2e1 influencia o comportamento exploratório, mas não é necessário para a diferenciação hormonal hipofisária no zebrafish (Danio rerio)." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/5/5135/tde-16082017-133351/.
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Hipopituitarismo congênito é caracterizado por deficiência hormonal múltipla devido a mutações de fatores de transcrição envolvidos na embriogênese hipofisária. As células-tronco estão presentes na hipófise e são caracterizadas por dar origem a uma célula progenitora e uma célula indiferenciada por divisão assimétrica. Estão envolvidas na hipófise em processos de alta demanda metabólica em diferentes fases da vida. Em estudos prévios, observou-se o acúmulo dos marcadores de células-tronco Sox2 e Nr2e1 no camundongo Ames, que apresenta mutação no gene Prop1. O Sox2 é o marcador consenso de células-tronco na hipófise enquanto que o Nr2e1, nunca antes caracterizado na hipófise, é essencial para a manutenção de células-tronco e neogenese no cérebro. A perda de função deste gene pode causar agressão e falta de instinto materno em camundongos. Com isso, o objetivo desse projeto foi utilizar o animal modelo zebrafish para avaliar o papel repressor do gene prop1 e caracterizar o gene nr2e1 bem como, confirmar se o mesmo está envolvido com a diferenciação terminal na hipófise, e sua interferência no comportamento do animal mutado. O zebrafish se encaixa adequadamente nesse projeto pois é de fácil manutenção, econômico e com rápido desenvolvimento. No presente estudo criou-se 2 modelos de zebrafish utilizando-se a técnica de edição genômica conhecida como CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) para nocautear os genes prop1 e nr2e1. Esta técnica permite uma interrupção específica e substituição de bases no genoma, resultando em uma alta especificidade, baixa toxicidade celular e é herdável. O zebrafish homozigoto com mutação no gene nr2e1 se desenvolve e reproduz como o animal controle, porém apresenta um comportamento mais exploratório quando comparado com o animal selvagem e o heterozigoto. A imunofluorescência para o anticorpo Sox2 no animal mutado mostrou se diferente do selvagem, pois apresenta um aumento da expressão temporal e o mesmo não se colocaliza com o Nr2e1. A imunofluorescência feita com os hormônios não se mostrou diferente entre o mutado e o selvagem. Conclui-se diante dos achados de normalidade do desenvolvimento, fertilidade, ausência de co-localização com o gene Sox2 e presença de hormônios como Tsh, Fsh e Gh, que o gene nr2e1 não é crucial na diferenciação terminal na hipófise porém o animal mutado apresenta um comportamento diferente do animal selvagem. Os resultados da caracterização do zebrafish com mutação no gene prop1 ainda estão em andamento devido a dificuldade de se estabelecer essa linhagemCongenital hypopituitarism is characterized by multiple hormone deficiencies due to mutations in transcription factors involved in pituitary embryogenesis. Stem cells, which by definition can each give rise to a progenitor and an undifferentiated cell by asymmetric division, are present in the pituitary gland and are important during periods of high metabolic demand in different phases of life. In previous studies, the accumulation of the stem cell markers Sox2 and Nr2e1 was observed in the Ames mouse, which harbors a mutation in Prop1. Sox2 is the consensus stem cell marker in the pituitary gland, while the role of Nr2e1 in the pituitary development has not been characterized although it is essential for neural stem cell maintenance and neogenesis in the brain and its loss of function causes pathological aggression and lack of maternal instinct in mice. In this project, the zebrafish animal model was used to characterize the role of nr2e1, to confirm whether this gene can be involved in the pituitary terminal differentiation, and to determine the effects of this gene on animal behavior. The zebrafish is a particularly appropriate model for use in this project because it is easy to maintain, is economical, and has a rapid metabolism and growth rate. In the present study, we created 2 zebrafish models by knocking out prop1 and nr2e1 using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome-editing technique. This technique enables highly specific gene/reading frame interruption and/or base substitution in the genome, with low cellular toxicity and high heritability. Zebrafish with homozygous nr2e1 mutations develop and reproduce similarly to wild-type zebrafish, but present a more exploratory behavioral pattern compared to wild-type and heterozygous zebrafish. Based on immunofluorescence, Sox2 expression was higher in the mutant zebrafish than in the wild type and was not co-localized with Nr2e1 expression. Hormone expression did not differ between wild-type and mutant zebrafish. We conclude that nr2e1 is not crucial in the terminal differentiation of the hormone-forming pituitary gland; however, it induces a distinct behavioral phenotype at the larval stage. Analyses of zebrafish harboring a prop1 mutation are ongoing owing to issues with the establishment of the lineage
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Zhu, Jiang. "PART I CRYSTAL STRUCTURE OF A DIMERIZATION DOMAIN OF DROSOPHILA CAPRIN. PART II CHARACTERIZATION OF TWO CAS13B CRISPR-CAS SYSTEMS FROM PORPHYROMONAS GINGIVALIS." OpenSIUC, 2018. https://opensiuc.lib.siu.edu/dissertations/1503.
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PART I: CRYSTAL STRUCTURE OF A DIMERIZATIO DOMAIN OF DROSOPHILA CAPRIN Drosophila Melanogaster Caprin (dCaprin) shares conversed HR1 domain with Caprin protein family members, which are RNA binding proteins that play critical roles in many important biological processes, such as synaptic plasticity, stress response, innate immune response and cellular proliferation. One of the Caprin protein family members, Caprin-1, is involved in the pathway of several human diseases, including breast cancer, neurodegenerative disorders, osteosarcoma, hearing loss, and viral infection. The functions of Caprin protein relies on their molecular interactions. Several direct interactions have been established between Caprin-1 and the Fragile X mental retardation protein (FMRP), Ras-GAP SH3 domain-binding protein 1 (G3BP1), and the Japanese encephalitis virus (JEV) core protein. We have determined the crystal structures of a fragment (residues 187-309) of Drosophila Melanogaster Caprin (dCaprin), which mediates homodimerization through a substantial interface created by a mainly alpha-helical fold. A larger hollow surface is created by homodimerization suggesting a protein binding groove. The FMRP binding should not affect dCaprin homodimerization for an integral alpha-helix in the dimeric dCaprein which formed by the FMRP interacting sequence motif. PART II: CHARACTERIZATION OF TWO TYPE VI-B CRESPR SYSTEMS: PGI5CAS1B AND PGI8CAS13B WHICH EFFECTOR PROTEINS ARE CAPABLE OF PROCESSING PRE-CRRNA INTO MATURE CRRNA CRISPR-Cas adaptive immune system protects microorganism from foreign nucleic acids invasion through endonucleases activity guided by RNA, which system has turned to a powerful genome editing tool applied to a multifold species, ranging from bacteria to human. Pgi5Cas13b and Pgi8Cas13b are identified by a computational sequence database mining approach, the CRISPR arrays lack of Cas1 and Cas2 encoding genes but contain a large candidate effector protein around 1,200 amino acids. They can be potentially classified as subtype VI-B CRISPR-Cas systems. We characterized the mature crRNA for Pgi5Cas13b and Pgi8Cas13b via Northern blot and small RNA sequencing. By EMSA (Electrophoretic mobility shift assay) experiments, we identified the binding constant between Pgi5Cas1b/Pgi8Cas13b and their corresponding crRNAs. The CRISPR loci of two of the Cas13b systems were cloned in pACYC vector and expressed in E. coli cells. Small RNAs were extracted and characterized by Northern Blotting and NGS (small RNA-seq) methods. The NGS results revealed the exact sequences of the crRNAs, which show a few new features not previously observed in other systems, including the longest spacer-derived sequences (32 and 31 nt), spacer-derived sequences flanking both ends of the full DR-derived sequence. The results also indicate different rules of pre-crRNA processing by the Pgi5Cas13b and Pgi8Cas13b systems. The characterization of these CRISPR systems extends the application of CRISPR based genome editing tools and promotes the development of single transcript manipulation tools.
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Chen, Hui-Min. "A More Accessible Drosophila Genome to Study Fly CNS Development: A Dissertation." eScholarship@UMMS, 2015. http://escholarship.umassmed.edu/gsbs_diss/758.
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Understanding the complex mechanisms to assemble a functional brain demands sophisticated experimental designs. Drosophila melanogaster, a model organism equipped with powerful genetic tools and evolutionarily conserved developmental programs, is ideal for such mechanistic studies. Valuable insights were learned from research in Drosophila ventral nerve cord, such as spatial patterning, temporal coding, and lineage diversification. However, the blueprint of Drosophila cerebrum development remains largely unknown.
Neural progenitor cells, called neuroblasts (NBs), serially and stereotypically produce neurons and glia in the Drosophila cerebrum. Neuroblasts inherit specific sets of early patterning genes, which likely determine their individual identities when neuroblasts delaminate from neuroectoderm. Unique neuroblasts may hence acquire the abilities to differentially interpret the temporal codes and deposit characteristic progeny lineages. We believe resolving this age-old speculation requires a tracing system that links patterning genes to neuroblasts and corresponding lineages, and further allows specific manipulations.
Using modern transgenic systems, one can immortalize transient NB gene expressions into continual labeling of their offspring. Having a collection of knockin drivers that capture endogenous gene expression patterns would open the door for tracing specific NBs and their progenies based on the combinatorial expression of various early patterning genes. Anticipating the need for a high throughput gene targeting system, we created Golic+ (gene targeting during oogenesis with lethality inhibitor and CRISPR/Cas “plus”), which features efficient homologous recombination in cystoblasts and a lethality selection for easy targeting candidate recovery. Using Golic+, we successfully generated T2AGal4 knock-ins for 6 representative early patterning genes, including lab, unpg, hkb, vnd, ind, and msh. They faithfully recapitulated the expression patterns of the targeted genes. After preserving initial NB expressions by triggering irreversible genetic labeling, we revealed the lineages founded by the NBs expressing a particular early patterning gene.
Identifying the neuroblasts and lineages that express a particular early patterning gene should elucidate the genetic origin of neuroblast diversity. We believe such an effort will lead to a deeper understanding of brain development and evolution.
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Hermanns, Veronica Verfasser], Rolf [Akademischer Betreuer] Wagner, and William F. [Akademischer Betreuer] [Martin. "Untersuchungen zur Funktion des CRISPR/Cas-Systems von E. coli: Isolierung und Charakterisierung spezifischer Komponenten des Cascade-Komplexes und Aufbau eines Systems zur Spacer Integration / Veronica Hermanns. Gutachter: William Martin. Betreuer: Rolf Wagner." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2014. http://d-nb.info/1053761597/34.
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Santos, Rafael Miyashiro Nunes dos. "Substituição gênica ortotópica de porco para humano baseada em CRISPR/Cas9 e recombinases para xenotransplante." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/5/5168/tde-14112017-153947/.
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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 SuínosHumanized 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
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Stens, Cassandra, Isabella Enoksson, and Sara Berggren. "The CRISPR-Cas system." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-171997.
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Derived from and inspired by the adaptive immune system of bacteria, CRISPR has gone from basic biology knowledge to a revolutionizing biotechnological tool, applicable in many research areas such as medicine, industry and agriculture. The full mechanism of CRISPR-Cas9 was first published in 2012 and various CRISPR-Cas systems have already passed the first stages of clinical trials as new gene therapies. The immense research has resulted in continuously growing knowledge of CRISPR systems and the technique seems to have the potential to greatly impact all life on our planet. Therefore, this literature study aims to thoroughly describe the CRISPR-Cas system, and further suggest an undergraduate laboratory exercise involving gene editing with the CRISPR-Cas9 tool. In this paper, we describe the fundamental technical background of the CRISPR-Cas system, especially emphasizing the most studied CRISPR-Cas9 system, its development and applications areas, as well as highlighting its current limitations and ethical concerns. The history of genetic engineering and the discovery of the CRISPR system is also described, along with a comparison with other established gene editing techniques. This study concludes that a deeper knowledge about CRISPR is important and required since the technique is applicable in many research areas. A laboratory exercise will not only inspire but also provide extended theoretical and practical knowledge for undergraduate students.
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Hedberg, Rickard. "Preimplantation genetic diagnosis and therapy in humans- Opportunities and risks." Thesis, Örebro universitet, Institutionen för medicinska vetenskaper, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-81532.
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IntroductionPreimplantation Genetic Diagnosis (PGD) was developed in the 1990s and has been used since to diagnose and discard embryos with genetic conditions or chromosomal abnormalities. CRISPR-Cas9 was discovered in 2012 and has been used in research, but has not become clinical practice on humans yet. CRISPR-Cas9 could potentially be applied to treat and prevent genetic disorders.AimThe aim was to investigate the ethical dilemmas of each method through a set of research questions. The ethics of applying PGD according to Swedish guidelines and applying CRISPR-Cas9 on humans was investigated.MethodologyThis was not a systematic literature review. Instead, articles have been selected based on their explanation of each method and uniqueness or volume of ethical arguments surrounding each method, that is of relevance for the discussed issues.ResultsArguments in favour of PGD addressed among other things the somatic and psychological health of future children and parents along with the economical benefits. Arguments against PGD addressed different dilemmas of discarding an embryo and thereby a future individual. Arguments against CRISPR-Cas9 addressed technical limitations, our limited knowledge of genetics and more. Arguments in favour addressed benefits in clinical medicine and research.ConclusionsPGD according to Swedish guidelines was found to be ethically acceptable, since its restrictive use that have not given room for ethically dubious applications. CRISPR-Cas9 was found not to be safe enough for human applications at this moment due to technical limitations. If these were to be solved, caution and restraint must be urged.
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Wong, Shi Pey. "Analysis of the adaptation mechanism in the type II-A CRISPR-Cas system." Doctoral thesis, Humboldt-Universität zu Berlin, 2019. http://dx.doi.org/10.18452/19806.
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Das RNA-guided adaptive Immunsystem CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) immunisiert prokaryotische Zellen gegenüber mobilen genetischen Elementen (MGEs). Bei der Adaption wird eine kurze Nukleinsäurensequenz (prespacer) von den MGEs gewonnen, verarbeitet und schließlich als spacer in das CRISPR-Array integriert. Cas1 und Cas2, die Hauptbestandteile der Adaption, bilden einen Integrase-Komplex, welcher neue spacer in das CRISPR-Array integriert. Der molekulare Mechanismus für die Adaptiondes Typ II-A Systems, welches cas9, cas1, cas2, csn2 und tracrRNA codiert, ist bis heute nicht vollständig verstanden. Daher untersuchten wir die Anforderungen der verschiedenen Cas-Proteine für den Adaptionsprozess.
Wir verifizierten die Adaptions-Aktivität von Typ II-A Systemen des Streptococcus thermophilus LMD-9 anhand von Adaptionsstudien nach Phagen-Infektion. Dabei beobachteten wir höhere Akquisitionsraten im CRISPR3-Lokus im Vergleich zum CRISPR1-Lokus. Unsere Plasmid-basierte Adaptionsstudie bestätigte die Notwendigkeit von Cas9, zusätzlich zu Cas1, Cas2 und Csn2 bei der Adaption. Der yeast two-hybrid und der pull-down Ansatz zeigten sowohl spezifische Interaktionen zwischen den Cas-Proteinen, als auch Interaktionen zwischen Cas-Proteinen sowie DNA-Reparatur Proteinen. Die Regionen der Cas1 und Cas9 Interaktion wurden durch SPOT peptide assay identifiziert. Zusammenfassend weist unsere Studie darauf hin, dass Cas-Proteine sowohl mit Proteinen innerhalb, als auch außerhalb des CRISPR-Cas Systems interagieren, und bietet somit eine Basis für die Erforschung der möglichen Funktionen von DNA-Reparatur Proteinen in CRISPR-Cas Systemen und vice versa.The RNA guided adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats) Cas (CRISPR-associated) immunizes prokaryotic cells against mobile genetic elements (MGEs). During spacer acquisition stage, a short nucleic acid sequence (prespacer) is acquired from the MGEs, processed and finally integrated into the CRISPR array as a spacer, which serves as genetic memory to defend against the invasion of the cognate MGEs. The molecular mechanism for the spacer acquisition of the type II A systems, which encode cas9, cas1, cas2, csn2 and tracrRNA, is still not fully understood. Therefore, we investigated the requirement of the different Cas proteins for spacer acquisition. We verified the acquisition activity of the type II A systems of Streptococcus thermophilus LMD 9 via spacer acquisition studies by phage challenge. We observed higher acquisition rates in the CRISPR3 locus compared to the CRISPR1 locus. Our plasmid-based spacer acquisition study confirmed in addition to Cas1, Cas2 and Csn2 the requirement of Cas9 for spacer acquisition. Yeast two hybrid and pull down approaches revealed specific interactions among the Cas proteins, as well as interactions between Cas and DNA repair proteins. The interaction regions of Cas1 with Cas9 were identified by SPOT peptide assay. Altogether, our study suggests that Cas proteins interact with proteins within and beyond the CRISPR Cas systems, and it provides a basis for the investigation of the potential roles of DNA repair proteins in the CRISPR Cas systems and/or vice versa.
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Lin, ChieYu. "Characterization and Optimization of the CRISPR/Cas System for Applications in Genome Engineering." Thesis, Harvard University, 2014. http://etds.lib.harvard.edu/hms/admin/view/61.
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The ability to precisely manipulate the genome in a targeted manner is fundamental to driving both basic science research and development of medical therapeutics. Until recently, this has been primarily achieved through coupling of a nuclease domain with customizable protein modules that recognize DNA in a sequence-specific manner such as zinc finger or transcription activator-like effector domains. Though these approaches have allowed unprecedented precision in manipulating the genome, in practice they have been limited by the reproducibility, predictability, and specificity of targeted cleavage, all of which are partially attributable to the nature of protein-mediated DNA sequence recognition. It has been recently shown that the microbial CRISPR-Cas system can be adapted for eukaryotic genome editing. Cas9, an RNA-guided DNA endonuclease, is directed by a 20-nt guide sequence via Watson-Crick base-pairing to its genomic target. Cas9 subsequently induces a double-stranded DNA break that results in targeted gene disruption through non-homologous end-joining repair or gene replacement via homologous recombination. Finally, the RNA guide and protein nuclease dual component system allows simultaneous delivery of multiple guide RNAs (sgRNA) to achieve multiplex genome editing with ease and efficiency.
The potential effects of off-target genomic modification represent a significant caveat to genome editing approaches in both research and therapeutic applications. Prior work from our lab and others has shown that Cas9 can tolerate some degree of mismatch with the guide RNA to target DNA base pairing. To increase substrate specificity, we devised a technique that uses a Cas9 nickase mutant with appropriately paired guide RNAs to efficiently inducing double-stranded breaks via simultaneous nicks on both strands of target DNA. As single-stranded nicks are repaired with high fidelity, targeted genome modification only occurs when the two opposite-strand nicks are closely spaced. This double nickase approach allows for marked reduction of off-target genome modification while maintaining robust on-target cleavage efficiency, making a significant step towards addressing one of the primary concerns regarding the use of genome editing technologies.
The ability to multiplex genome engineering by simply co-delivering multiple sgRNAs is a versatile property unique to the CRISPR-Cas system. While co-transfection of multiple guides is readily feasible in tissue culture, many in vivo and therapeutic applications would benefit from a compact, single vector system that would allow robust and reproducible multiplex editing. To achieve this, we first generated and functionally validated alternate sgRNA architectures to characterize the structure-function relationship of the Cas9 protein with the sgRNA in DNA recognition and cleavage. We then applied this knowledge towards the development and optimization of a tandem synthetic guide RNA (tsgRNA) scaffold that allows for a single promoter to drive expression of a single RNA transcript encoding two sgRNAs, which are subsequently processed into individual active sgRNAs.
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Maikova, Anna. "The CRISPR-Cas system of human pathogen Clostridium difficile : function and regulation." Thesis, Université de Paris (2019-....), 2019. http://www.theses.fr/2019UNIP7091.
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Clostridium difficile (nouveau nom Clostridioides difficile) est une bactérie à Gram-positif, sporulante, anaérobie stricte, présente dans le sol et les environnements aquatiques, ainsi que dans le tractus intestinal des mammifères. C. difficile est l’un des principaux clostridies pathogènes. Cette bactérie est devenue un vrai problème de santé publique associé à l'antibiothérapie dans les pays industrialisés. La diarrhée associée à C. difficile est actuellement la diarrhée nosocomiale la plus fréquente en Europe et dans le monde. Depuis la dernière décennie, la proportion de formes d’infections graves a augmentée en raison de l’émergence des souches hypervirulantes et épidémiques comme la souche R20291 de ribotype 027. L’infection à C. difficile provoque la diarrhée, la colite et même la mort. De nombreux aspects de la pathogenèse de C. difficile restent mal compris. En particulier, les mécanismes moléculaires de son adaptation aux conditions changeantes de l'hôte doivent être examinés.Durant le cycle d'infection, C. difficile survit dans des communautés intestinales riches en bactériophages, en utilisant des systèmes qui contrôlent les échanges génétiques favorisés dans ces environnements complexes. Au cours de la dernière décennie, les systèmes CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (associés aux CRISPR) d'immunité adaptative chez les procaryotes contre des éléments génétiques exogènes sont devenus le centre d'intérêt scientifique parmi les divers systèmes de défense bactérienne.Des études antérieures ont révélé la présence d'ARN CRISPR abondants chez C. difficile. Cette bactérie possède un système CRISPR original, caractérisé par la présence d'un grand nombre de cassettes CRISPR (12 dans la souche 630 et 9 dans la souche hypervirulante R20291), de deux ou trois opérons cas conservés dans la majorité des génomes séquencés de C. difficile et la localisation au sein des prophages de plusieurs cassettes CRISPR. Cependant, le rôle de CRISPR-Cas dans la physiologie et le cycle infectieux de cet important pathogène reste obscur.Les objectifs de ce travail sont les suivants:1) étudier le rôle et la fonctionnalité du système CRISPR-Cas de C. difficile dans les interactions avec des éléments d'ADN étrangers (tels que les plasmides), 2) révéler la manière dont le système CRISPR-Cas de C. difficile est régulé et fonctionne dans des conditions de culture bactérienne différentes, incluant la réponse aux stress.Dans la présente thèse, la fonctionnalité du système CRISPR-Cas de C. difficile a été étudiée (chapitre 2). Grâce à des tests d'efficacité de conjugaison, la fonction défensive (en interférence) du système CRISPR-Cas a été démontrée. La corrélation entre les niveaux d'expression des ARN CRISPR et les niveaux d'interférence observés a également été montrée. De plus, grâce à la série d’expériences d’interférence, la fonctionnalité des motifs PAM (protospacer adjacent motifs) a été confirmée en accord avec des prédictions in silico. Le consensus fonctionnel de PAM a été déterminé expérimentalement avec les bibliothèques des plasmides. La fonction adaptative du système CRISPR-Cas de C. difficile a été également démontrée pour la souche de laboratoire. Le rôle de plusieurs opérons cas dans la fonctionnalité du système CRISPR de C. difficile est démontré aussi dans ce chapitre.Le chapitre 3 montre le lien entre le système CRISPR-Cas et un nouveau système toxine-antitoxine de type I, ainsi que leur possible co-régulation dans des conditions de biofilm et de stress. Ce chapitre définit également le rôle possible du c-di-GMP dans la régulation du système CRISPR-Cas de C. difficile. De plus, le chapitre 4 décrit l'utilisation du système CRISPR-Cas endogène comme nouvel outil pour la rédaction du génome de C. difficile.En conclusion, les données obtenues mettent en évidence les caractéristiques originales du système CRISPR-Cas actif de C. difficile et démontrent son potentiel biotechnologiqueClostridium difficile (the novel name – Clostridioides difficile) is a Gram-positive, strictly anaerobic spore forming bacterium, found in soil and aquatic environments as well as in mammalian intestinal tracts. C. difficile is one of the major pathogenic clostridia. This bacterium has become a key public health issue associated with antibiotic therapy in industrialized countries. C. difficile-associated diarrhoea is currently the most frequently occurring nosocomial diarrhoea in Europe and worldwide. Since the last decade the number of severe infection forms has been rising due to emergence of the hypervirulent and epidemic strains as ribotype 027 R20291 strain. C. difficile infection causes diarrhoea, colitis and even death. Many aspects of C. difficile pathogenesis remain poorly understood. Particularly, the molecular mechanisms of its adaptation to changing conditions inside the host are to be scrutinized. During the infection cycle C. difficile survives in bacteriophage-rich gut communities possibly by relying on some special systems that control the genetic exchanges favored within these complex environments. During the last decade, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems of adaptive prokaryotic immunity against exogenic genetic elements has become the center of interest among various anti-invader bacterial defense systems.Previous studies revealed the presence of abundant and diverse CRISPR RNAs in C. difficile. C. difficile has an original CRISPR system, which is characterized by the presence of an unusually large set of CRISPR arrays (12 arrays in the laboratory 630 strain and 9 ones in the hypervirulent R20291 strain), of two or three sets of cas genes conserved in the majority of sequenced C. difficile genomes and the prophage location of several CRISPR arrays. However, the role CRISPR-Cas plays in the physiology and infectious cycle of this important pathogen remains obscure.The general aims of this work run as follows: 1) to investigate the role and the functionality of C. difficile CRISPR-Cas system in the interactions with foreign DNA elements (such as plasmids), 2) to reveal the way C. difficile CRISPR-Cas system expression is regulated and functions in different states of bacterial culture, including its response to stresses. In the present PhD thesis the functionality of C. difficile CRISPR-Cas system was investigated (Chapter 2). Through conjugation efficiency assays defensive function (in interference) of C. difficile CRISPR-Cas system was demonstrated. The correlation between the previously known levels of expression of CRISPR RNAs and the observed levels of interference has also been shown. Moreover, through the series of interference experiments the functionality of PAMs (protospacer adjacent motifs) was confirmed, which have already been predicted in silico. Additionally, the general functional PAM consensus was determined using PAM libraries experiments. Furthermore, an adaptive function of C. difficile CRISPR-Cas system was shown for laboratory strain. The role of multiple cas operons in C. difficile CRISPR functionality is also demonstrated in this Chapter.In Chapter 3 the link between C. difficile CRISPR-Cas system and a new type I toxin-antitoxin system is demonstrated, as well as a possible co-regulation under biofilm and stress conditions of CRISPR-Cas system and these toxin-antitoxin modules. This Chapter also defines a possible role of c-di-GMP in regulation of C. difficile CRISPR-Cas system. Additionally, Chapter 4 describes the utilization of endogenous C. difficile CRISPR-Cas system as a novel tool for genome editing in C. difficile. Altogether, the obtained data highlight the original features of active C. difficile CRISPR-Cas system and demonstrate its biotechnological potential
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Yokooji, Yusuke. "Genetic studies on the metabolism and CRISPR-Cas system of Thermococcus kodakarensis." 京都大学 (Kyoto University), 2013. http://hdl.handle.net/2433/180506.
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Amlinger, Lina. "The type I-E CRISPR-Cas system : Biology and applications of an adaptive immune system in bacteria." Doctoral thesis, Uppsala universitet, Mikrobiologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-312234.
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CRISPR-Cas systems are adaptive immune systems in bacteria and archaea, consisting of a clustered regularly interspaced short palindromic repeats (CRISPR) array and CRISPR associated (Cas) proteins. In this work, the type I-E CRISPR-Cas system of Escherichia coli was studied. CRISPR-Cas immunity is divided into three stages. In the first stage, adaptation, Cas1 and Cas2 store memory of invaders in the CRISPR array as short intervening sequences, called spacers. During the expression stage, the array is transcribed, and subsequently processed into small CRISPR RNAs (crRNA), each consisting of one spacer and one repeat. The crRNAs are bound by the Cascade multi-protein complex. During the interference step, Cascade searches for DNA molecules complementary to the crRNA spacer. When a match is found, the target DNA is degraded by the recruited Cas3 nuclease. Host factors required for integration of new spacers into the CRISPR array were first investigated. Deleting recD, involved in DNA repair, abolished memory formation by reducing the concentration of the Cas1-Cas2 expression plasmid, leading to decreased amounts of Cas1 to levels likely insufficient for spacer integration. Deletion of RecD has an indirect effect on adaptation. To facilitate detection of adaptation, a sensitive fluorescent reporter was developed where an out-of-frame yfp reporter gene is moved into frame when a new spacer is integrated, enabling fluorescent detection of adaptation. Integration can be detected in single cells by a variety of fluorescence-based methods. A second aspect of this thesis aimed at investigating spacer elements affecting target interference. Spacers with predicted secondary structures in the crRNA impaired the ability of the CRISPR-Cas system to prevent transformation of targeted plasmids. Lastly, in absence of Cas3, Cascade was successfully used to inhibit transcription of specific genes by preventing RNA polymerase access to the promoter. The CRISPR-Cas field has seen rapid development since the first demonstration of immunity almost ten years ago. However, much research remains to fully understand these interesting adaptive immune systems and the research presented here increases our understanding of the type I-E CRISPR-Cas system.
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Stachler, Aris-Edda [Verfasser]. "Das CRISPR-Cas-System von Haloferax volcanii: CRISPRi und Autoimmunität / Aris-Edda Stachler." Ulm : Universität Ulm, 2017. http://d-nb.info/1140118145/34.
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Maier, Lisa-Katharina [Verfasser]. "Das CRISPR-Cas-System von Haloferax volcanii - Voraussetzungen für eine funktionelle Interferenzreaktion / Lisa-Katharina Maier." Ulm : Universität Ulm. Fakultät für Naturwissenschaften, 2015. http://d-nb.info/1075253039/34.
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Zöphel, Judith [Verfasser], and Lennart [Akademischer Betreuer] Randau. "Characterization of a type I-B CRISPR-Cas system of Clostridium thermocellum / Judith Zöphel. Betreuer: Lennart Randau." Marburg : Philipps-Universität Marburg, 2016. http://d-nb.info/1089078226/34.
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Özcan, Ahsen [Verfasser], and Kai [Akademischer Betreuer] Papenfort. "Characterization of the type IV CRISPR-Cas system of aromatoleum aromaticum EbN1 / Ahsen Özcan ; Betreuer: Kai Papenfort." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1196009368/34.
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Hopes, Amanda. "Expanding the molecular toolbox in diatoms : developing a transformation system, CRISPR-Cas and Inverse Yeast-1-hybrid." Thesis, University of East Anglia, 2017. https://ueaeprints.uea.ac.uk/66542/.
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Diatoms are single celled microalgae with intricately patterned silica cell walls. This cosmopolitan group is a dominant primary producer with many species playing key roles in marine, estuarine and freshwater habitats. Furthermore, due to their silica frustule, lipid production and a range of other chemical and physiological adaptations, diatoms have high potential for biotechnology. Despite their diversity and ecological relevance, molecular tools for diatoms are often underrepresented and limited to a small number of species. This PhD expands the molecular toolbox for two key species: Thalassiosira pseudonana, a model, centric, temperate diatom with a heavily silicified frustule and Fragilariopsis cylindrus, a key, pennate diatom in marine psychrophilic waters and sea-ice. A transformation system has been developed in F. cylindrus leading to the expression of egfp and shble transgenes under the control of an endogenous FCP promoter. This method has been applied to understanding the role of the SITMyb gene, a potential transcription factor with links to silica metabolism, by overexpression. In-silico and in-vitro modelling of the SITMyb gene has been performed and preliminary development of an inverse yeast-1-hybrid system, to elucidate potential transcription factor binding sites, has been carried out. F. cylindrus is the first genetically tractable polar microalgae and appears to be the first psychrophilic eukaryote to be transformed. CRISPR-Cas is a targeted genome editing tool, fast becoming an essential method in any molecular toolbox. This thesis demonstrates development in T. pseudonana by successfully editing the urease gene through a programmed deletion using two sgRNAs. As a model diatom, several molecular tools are already available for T. pseudonana, however this is the first time a targeted knock-out has been achieved in this species. In addition Golden-Gate cloning has been used to produce the construct, giving this method a large degree of flexibility and future potential for multiplexing.
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Martínez, Fernández Carmen 1993. "C elegans and CRISPR/Cas gene editing to study BAP1 cancer-related mutations and cisplatin chemoresistance." Doctoral thesis, TDX (Tesis Doctorals en Xarxa), 2021. http://hdl.handle.net/10803/671159.
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Model organisms and gene-editing strategies are fundamental to
address a variety of scientific questions from basic science to
biomedical research. Here, we reinforced the use of two powerful
tools, the experimental system Caenorhabditis elegans and the
CRISPR/Cas gene-editing technology, to model cancer-related
mutations and investigate cisplatin-based chemoresistance.
We have established a model to study BAP1 cancer predisposition
syndrome-related mutations in the BAP1 ortholog ubh-4. By
exploring distinct ubh-4 alleles, we have discovered a synthetic
interaction between ubh-4 and rpn-9, an essential regulatory subunit
involved in proteasome assembly. Moreover, we suggest a
cooperating role between these genes in the ubiquitin-mediated
proteostasis at meiotic prophase.
In addition, we have exploited C. elegans to study the toxicity of
cisplatin-based therapies in different ways. First, by studying the
impact of glucose and lipid metabolism on cisplatin toxicity. Then,
we have described the harmful effect of cisplatin in mitochondrial
functions. Finally, we have established a method to investigate the
cisplatin-induced neurotoxicity by using an automated worm tracking
system and discovered a protective role of dopamine.Los organismos modelo y las estrategias de edición genética son fundamentales para desentrañar incógnitas en ciencias de la vida, desde la investigación básica hasta investigación aplicada a la biomedicina. En este estudio, reafirmamos la importancia del uso de dos potentes herramientas, el sistema experimental Caenorhabditis elegans y la tecnología de edición genética CRISPR/Cas, para modelar mutaciones relacionadas con cáncer e investigar la quimiorresistencia al cisplatino. Hemos modelado mutaciones asociadas al síndrome de predisposición tumoral BAP1, en ubh-4/BAP1. Explorando el efecto de distintos alelos mutantes de ubh-4, hemos descubierto una interacción sintética entre ubh-4 y rpn-9, el cual codifica para una subunidad reguladora esencial para el ensamblaje del proteasoma. Además, proponemos que la cooperación funcional de dichos genes está implicada en la degradación de proteínas mediada por el sistema ubiquitina-proteasoma durante la profase meiótica. También hemos investigado la respuesta generada por la terapia con cisplatino en C. elegans. Por una parte, hemos demostrado que la toxicidad inducida por el cisplatino puede modularse alterando el metabolismo glucídico y lipídico. Por otro lado, hemos observado que esta droga genera disfunción mitocondrial. Finalmente, mediante un sistema automatizado, hemos puesto a punto un método para evaluar el efecto neurotóxico del cisplatino en el nemátodo y hemos encontrado que la dopamina posee un efecto protector.
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Millet, Jonathan. "Stratégies d'analyse spatio-temporelle de l‟épissage alternatif chez Caenorhabditis elegans." Thesis, Bordeaux, 2015. http://www.theses.fr/2015BORD0437/document.
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L‟épissage alternatif est un mécanisme de régulation de l‟expression des gènes ayant pris une importance croissante dans l‟étude du vivant. Si des méthodes existent pour déterminer les gènes qui y sont soumis, peu d‟outils sont disponibles pour suivre ces événements d‟épissage in vivo au cours du développement. Pourtant, la caractérisation des régulations sous-jacentes à ces évènements et la détermination des facteurs impliqués sont dépendantes de stratégies fiables pour les visualiser dans des conditions physiologiques.Nous avons développé un système adapté à l‟étude d‟événements d‟épissage basé sur un rapporteur fluorescent bicolore. Nous l‟avons appliqué à cinq gènes de l‟organisme modèle Caenorhabditis elegans et avons suivi leur épissage in vivo.Parmi les différents gènes suivis, deux d‟entre eux suivaient un modèle d‟épissage potentiellement stochastique, un autre une absence d‟épissage alternatif détectable. Les deux derniers gènes présentent un profil d‟épissage spécifique à certain types cellulaires mais ont un effet toxique sur l‟organisme lorsque nous les avons exprimés à partir de concatémères extrachromosomiques. Pour remédier à cela, nous avons choisi de mettre en place une méthode simplifiée d‟insertion en simple copie des rapporteurs utilisant le CRISPR-Cas.Nos résultats indiquent que le système rapporteur fonctionne avec succès. Cependant, il peut encore être amélioré pour se rapprocher des taux physiologiques de transcription grâce à une insertion en simple copie dans le génome de l‟organisme. Nous avons également révélé un événement sous le contrôle de régulations spatiales, temporelles et conditionnelles. De plus, nous avons créé une série de constructions capables de déterminer les éléments en cis impliqués dans la régulation du gène top-1Alternative splicing is a regulatory mechanism of gene expression which is increasingly studied in Life Science. Methods exist to study this mechanism but specific tools to follow each alternative splicing event in a spatio-temporal manner are lacking. Yet, the characterization of the regulation and the elements that determines them depends on valide strategies for visualising them in physiological conditions.We have developped a dual-fluorescent reporter-based system in order to follow alternative splicing event regulation in vivo. It has been applied to five different genes in the model organism Caenorhabditis elegans. Among the genes followed, two follow a potentially stochastic scheme, one show no visible sign of alternative splicing. The last display tissue specific splicing patterns but developed a toxic effect in the animal when expressed from a multicopy extrachromosomal array. To remediate this problem, we decided to develop a method that allows for simpler single copy insertion of fluorescent reporter using CRISPR-Cas.Our results indicates that the dual-fluorescent reporter works well. However, this system can be upgraded by getting close to physiological rates of transcription allowed by single-copy insertion in the genome of C.elegans. We also discovered an alternatiove splicing event which follows a spatial, temporal and conditionnal regulation. Moreover, we constructed a set of different reporter to unravel the regulation observed in the gene top-1
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Dwarakanath, Srivatsa [Verfasser], and Lennart [Akademischer Betreuer] Randau. "Characterization of a minimal Type I CRISPR-Cas system found in Shewanella putrefaciens CN-32 / Srivatsa Dwarakanath. Betreuer: Lennart Randau." Marburg : Philipps-Universität Marburg, 2016. http://d-nb.info/1097534200/34.
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Müller-Esparza, Hanna [Verfasser], and Lennart [Akademischer Betreuer] Randau. "Characterization of DNA interference by a minimal Type I-F CRISPR-Cas system / Hanna Constanza Müller Esparza ; Betreuer: Lennart Randau." Marburg : Philipps-Universität Marburg, 2020. http://d-nb.info/1211086283/34.
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Müller, Esparza Hanna Constanza [Verfasser], and Lennart [Akademischer Betreuer] Randau. "Characterization of DNA interference by a minimal Type I-F CRISPR-Cas system / Hanna Constanza Müller Esparza ; Betreuer: Lennart Randau." Marburg : Philipps-Universität Marburg, 2020. http://d-nb.info/1211086283/34.
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Gebler, Christina [Verfasser], Frank [Gutachter] Buchholz, and Axel [Gutachter] Roers. "Developing the CRISPR/Cas-system for Inactivation of Proto-oncogenes in Human Cancer Cells / Christina Gebler ; Gutachter: Frank Buchholz, Axel Roers." Dresden : Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://d-nb.info/1227196482/34.
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Sokolowski, Richard D. "CRISPR RNA biogenesis by a Cas6 nuclease." Thesis, University of St Andrews, 2015. http://hdl.handle.net/10023/6861.
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Clustered regularly interspaced short palindromic repeats (CRISPRs) and associated (Cas) proteins form the basis of a prokaryotic adaptive immune system. Acquired sections of viral DNA are stored within the host genome as ‘spacers' flanked by ‘repeat' sequences. The CRISPR arrays are transcribed and processed to release mature CRISPR RNAs (crRNAs) – containing a single, intact spacer sequence – that are used by effector complexes to base-pair with matching hostile genetic elements and silence future infections. crRNA-biogenesis is thus an essential step within the defence pathway. Within Type I and III systems, the primary processing of the CRISPR transcript at repeat sites is performed almost exclusively by the CRISPR-specific riboendonuclease, Cas6. This thesis seeks to probe the catalytic mechanism of a Cas6 enzyme from the crenarchaeon Sulfolobus solfataricus (sso). Despite analogous generation of crRNA, ssoCas6 paralogues differ from previously characterised Cas6 examples in their lack of a canonical active site histidine residue. The work here builds on recent crystallographic evidence that the ssoCas6-1 paralogue unexpectedly adopts a dimeric conformation (PDB 3ZFV, 4ILR), to show that not only is the ssoCas6-1 dimer stable in solution but that this atypical arrangement is important to the activity of this particular enzyme. Furthermore, the ssoCas6-1 paralogue is shown to be the first in this family of endonucleases to employ multiple-turnover kinetics. The widespread diversity in Cas6 catalytic mechanisms reflects the plastic nature of the Cas6 active site and rapid co-evolution with substrate repeat sequences. The CRISPR/Cas environment within S. solfataricus is highly complex, containing three co-existing system types (Type I-A, III-A, III-B), five Cas6 paralogues and two families of CRISPR loci (AB and CD) that differ by repeat sequence. By probing the activity of an additional ssoCas6 paralogue (ssoCas6-3), which reveals different substrate specificities to those of ssoCas6-1, evidence emerges for functional coupling between ssoCas6 paralogues and downstream effector complexes, sufficient to regulate crRNA uptake and possibly even complex assembly.
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Wong, Shi Pey [Verfasser], Dina [Gutachter] Grohmann, Anita [Gutachter] Marchfelder, and Emmanuelle [Gutachter] Charpentier. "Analysis of the adaptation mechanism in the type II-A CRISPR-Cas system / Shi Pey Wong ; Gutachter: Dina Grohmann, Anita Marchfelder, Emmanuelle Charpentier." Berlin : Humboldt-Universität zu Berlin, 2019. http://d-nb.info/1188712713/34.
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Wong, Shi Pey [Verfasser], Dina Gutachter] Grohmann, Anita [Gutachter] Marchfelder, and Emmanuelle [Gutachter] [Charpentier. "Analysis of the adaptation mechanism in the type II-A CRISPR-Cas system / Shi Pey Wong ; Gutachter: Dina Grohmann, Anita Marchfelder, Emmanuelle Charpentier." Berlin : Humboldt-Universität zu Berlin, 2019. http://nbn-resolving.de/urn:nbn:de:kobv:11-110-18452/20603-1.
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Kollasser, Jana Verfasser], Theresia [Akademischer Betreuer] [Stradal, and Reinhard [Akademischer Betreuer] Köster. "Mutation and/or inactivation of actinregulatory genes relevant for infection using the CRISPR/Cas system in tissue cultured cells / Jana Kollasser ; Theresia Stradal, Reinhard Köster." Braunschweig : Technische Universität Braunschweig, 2019. http://d-nb.info/1188810537/34.
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Hagmann, Hanns Antony [Verfasser], Christoph U. [Gutachter] Schoen, Jörg [Gutachter] Vogel, Roy [Gutachter] Gross, and Johannes G. [Gutachter] Liese. "The impact of the CRISPR/Cas system on the interaction of Neisseria meningitidis with human host cells / Hanns Antony Hagmann ; Gutachter: Christoph U. Schoen, Jörg Vogel, Roy Gross, Johannes G. Liese." Würzburg : Universität Würzburg, 2020. http://d-nb.info/1204366810/34.
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Rigiracciolo, Damiano Cosimo, Sebastiano Andò, and Marcello Maggiolini. "Targeting systems vulnerabilities in uveal melanoma by CRISPR Cas/9 focal adhesion kinase (FAK) genome editing and therapeutic inhibition." Thesis, 2018. http://hdl.handle.net/10955/1843.
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Dottorato di Ricerca in Medicina Traslazionale. Ciclo XXXIl Melanoma Uveale rappresenta la neoplasia intraoculare più frequente nell’età adulta. Colpisce circa 2,500 individui ogni anno negli USA ed il 50% dei pazienti affetti da tale neoplasia sviluppa metastasi entro 5 anni dalla diagnosi. Non essendo state ancora identificate terapie efficaci, la sopravvivenza in presenza di metastasi è di circa 6 mesi. Il Melanoma Uveale è geneticamente caratterizzato dalla presenza di mutazioni somatiche attivanti a carico degli oncogeni GNAQ e GNA11, che codificano per due diverse subunità α delle proteine G. Tali mutazioni sono state identificate rispettivamente in circa il 94% dei casi di Melanoma Cutaneo ed il 4% dei casi di Melanoma Uveale. Sulla base di tali osservazioni, nel presente lavoro di tesi è stato valutato il ruolo esercitato da una proteina citoplasmatica ad attività tirosin-chinasica associata ai recettori per le integrine denominata FAK (focal adhesion kinase), nella progressione del Melanoma Uveale, sia in vitro che in vivo. In particolare, mediante analisi bioinformatica (www.cbioportal.com) delle alterazioni genomiche di campioni estratti da pazienti affetti da melanoma uveale (n=80), è stato inizialmente determinato che il gene codificante per FAK (PTK2) risulta over-espresso nel 56% dei casi. Inoltre, il presente studio condotto in cellule di Melanoma Uveale OMM1.3 (GNAQ/11 mutate) e in cellule ingegnerizzate per l’espressione di un recettore di membrana accoppiato a proteine-G (Gαq) attivato esclusivamente da ligandi sintetici denominate HEK293 DREADD/Gq, ha dimostrato il coinvolgimento di segnali mediati da GNAQ nell’attivazione di FAK attraverso il reclutamento del fattore coinvolto nello scambio di nucleotidi guaninici denominato TRIO e la proteina appartenente alla super-famiglia di Ras denominata Rho-A. A riprova, saggi biologici hanno dimostrato l’efficacia di specifici inibitori di FAK nei processi di proliferazione cellulare sia in cellule di Melanoma Uveale derivanti da lesioni primarie che da metastasi epatiche. Attraverso l’innovativo approccio genetico denominato CRISPR/Cas 9 genome editing (Clustered Regularly Interspaced Short Palindromic Repeats), il silenziamento dell’espressione di FAK ha ridotto significativamente la crescita del melanoma uveale in modelli sperimentali utilizzati in vivo. Collettivamente, i risultati ottenuti indicano che FAK può essere considerato un potenziale target terapeutico per il trattamento del Melanoma Uveale e di altre neoplasie caratterizzate da mutazioni oncogeniche a carico delle subunità αq/α11 dei recettori di membrana accoppiati a proteine G.
Università della Calabria
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Dambacher, Helena. "Die Etablierung des CRISPR/Cas9-Systems in humanen induzierten pluripotenten Stammzellen zur Untersuchung der Funktion des Kanalproteins Connexin 43 in der Embryonalentwicklung." Doctoral thesis, 2021. https://doi.org/10.25972/OPUS-24015.
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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 MutationenThe 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
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Eckermann, Kolja Neil. "Evaluation of genetic engineering and genome editing tools to develop multifactorial reproductive sterility or killing sperm systems for the improvement of the Sterile Insect Technique." Doctoral thesis, 2020. http://hdl.handle.net/21.11130/00-1735-0000-0005-14F7-E.
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Gawryszewska, Iwona. "Determinants of adaptation of Enterococcus faecalis and Enterococcus faecium to the hospital environment." Doctoral thesis, 2017. https://depotuw.ceon.pl/handle/item/2299.
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Enterococci are a part of the natural intestinal microbiota of humans and animals. They are also found in various environments such as food, water, sewage, soil and plants. Although enterococci are generally recognized as harmless bacteria, they are a common cause of hospital-associated infections in patients with risk factors. Among enterococci, Enterococcus faecalis and Enterococcus faecium represent two species of the highest clinical importance. Nosocomial isolates of E. faecalis and E. faecium predominantly belong to distinct enterococcal subpopulations, i.e. high-risk clones, which are almost exclusively identified in hospital settings.The objective of this PhD thesis was to investigate determinants of adaptation of E. faecalis and E. faecium to the hospital environment.Three groups of isolates were investigated: E. faecalis from hospital and non-hospital sources, E. faecalis and E. faecium isolated from invasive infections, the most dangerous and life threatening type of infections, and clinical isolates of E. faecalis and E. faecium with resistance to linezolid, one of the last resort agents in the treatment of enterococcal infections. The study showed that nosocomial E. faecalis and E. faecium isolates belonged to few high-risk clones and frequently displayed the phenotype of multidrug resistance. Hospital-associated isolates of E. faecalis were enriched in virulence factors and resistance determinants, frequently encoded on mobile genetic elements (MGEs). High-risk clones of E. faecalis were also characterized by a lower prevalence of CRISPR-Cas systems of bacterial defence against foreign DNA, such as plasmids or phages, in comparison to isolates from other sources. Among recent invasive enterococcal isolates, the percentage of E. faecium isolates was higher that it had been previously observed, presumably due to the increased prevalence of isolates of the lineage 78 belonging to high-risk clone CC17. This lineage was characterized by the presence of additional genomic regions, containing genes associated with various metabolic features. Linezolid resistance was observed mainly among E. faecium isolates caused by a mutation in the drug target (23S rRNA). Moreover, the plasmid-encoded ABC-type transporter OptrA, which extrudes the drug outside the cell was observed.In summary, adaptation of clinical isolates of E. faecalis and E. faecium to the hospital environment was determined by the acquisition of virulence factors, resistance determinants and genes encoding additional metabolic pathways as well as the loss of the CRISPR-Cas systems, which resulted in the emergence of multidrug-resistant high-risk clones. The ongoing evolution of one of E. faecium lineages is associated with the increasing role of this species in enterococcal infections.Enterokoki stanowią składnik naturalnej mikroflory jelitowej ludzi i zwierząt. Są one także obecne w różnych środowiskach, takich jak produkty spożywcze, woda, ścieki, gleba oraz rośliny. Mimo że enterokoki są powszechnie uznawane za nieszkodliwe, są one również częstą przyczyną zakażeń związanych z opieką zdrowotną u pacjentów obarczonych czynnikami ryzyka. Pośród enterokoków, Enterococcus faecalis i Enterococcus faecium stanowią dwa gatunki o największym znaczeniu klinicznym. Zdecydowana większość szpitalnych izolatów E. faecalis i E. faecium należy do odrębnych subpopulacji enterokoków, tak zwanych klonów wysokiego ryzyka, które są niemal wyłącznie identyfikowane w szpitalach.Celem rozprawy doktorskiej było ustalenie czynników adaptacji E. faecalis i E. faecium do środowiska szpitalnego.Do badań wykorzystano trzy grupy izolatów: szpitalne i pozaszpitalne izolaty E. faecalis, izolaty E. faecalis i E. faecium z zakażeń inwazyjnych, które stanowią najbardziej niebezpieczne i zagrażające życiu pacjentów zakażenia, oraz kliniczne izolaty E. faecalis i E. faecium wykazujące oporność na linezolid, antybiotyk ostatniego rzuty w leczeniu zakażeń enterokokowych.Badania wykazały, że szpitalne izolaty E. faecalis i E. faecium należały do kilku klonów wysokiego ryzyka oraz często wykazywały fenotyp wielolekooporności. Czynniki wirulencji oraz determinanty oporności, często kodowane na ruchomych elementach genetycznych, wykrywane były częściej w izolatach E. faecalis związanych ze środowiskiem szpitalnym niż pozaszpitalnym. Klony wysokiego ryzyka E. faecalis charakteryzowała również niska częstość występowania systemów CRISPR-Cas stanowiących bakteryjną ochronę przed obcym DNA, takim jak plazmidy czy fagi, w porównaniu z izolatami pochodzącymi z innych środowisk. Pośród inwazyjnych izolatów enterokoków pochodzących z ostatnich lat odsetek izolatów E. faecium był wyższy niż było to wcześniej obserwowane, prawdopodobnie ze względu na wyższą częstość występowania izolatów należących do linii 78 klonu wysokiego ryzyka CC17. Linia ta charakteryzowała się obecnością dodatkowych regionów w genomie, kodujących geny związane z różnymi cechami metabolicznymi. Fenotyp oporności na linezolid najczęściej występował wśród izolatów E. faecium oraz był związany z mutacją miejsca docelowego działania leku (cząsteczka 23S rRNA). Zaobserwowano także obecność kodowanego plazmidowo transportera typu ABC, OptrA, usuwającego lek z komórki.Podsumowywując, adaptacja klinicznych izolatów E. faecalis i E. faecium do środowiska szpitalnego była warunkowana przez nabycie czynników wirulencji, determinant oporności oraz genów kodujących dodatkowe szlaki metaboliczne, a także utratę systemów CRISPR-Cas, co doprowadziło do wyłonieniem się wielolekoopornych klonów wysokiego ryzyka. Trwająca ewolucja jednej z lini E. faecium wpływa na wzrost roli tego gatunku w infekcjach enterokokowych.
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Li, Hsin-Yu, and 李馨宇. "Characterization of CRISPR/Cas system in Klebsiella pneumoniae isolates." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/46437j.
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Halpin-Healy, Tyler Sheehan. "Structure and Function of a Transposon-Encoded CRISPR-Cas System." Thesis, 2021. https://doi.org/10.7916/d8-k048-fn54.
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CRISPR-Cas defense systems are employed by their hosts to prevent parasitization by mobile genetic elements. The discovery of nuclease-deficient CRISPR-Cas systems contained within transposon ends suggested a repurposing of the contained defense system. One such Type I-F3 CRISPR-Cas system was found inside Tn6677, a Tn7-like transposon within the genome of a Vibrio cholerae strain. Tn6677 requires coordination between the contained CRISPR-Cas system and the transposition proteins for effective transposition. Isolation of this system, and reduction to its minimal components, enabled RNA-guided integration of donor DNA in Escherichia coli. Base-pairing interactions between the user-specified CRISPR RNA and the target sequence precede the integration of donor DNA approximately 49-bp downstream of the end of the target sequence. This system is specific regardless of the supplied RNA guide, and successfully integrates donors of different lengths. The donor DNA is indicated by flanking cognate transposon end sequences. While clearly functional, the mechanism by which the transposition proteins and the CRISPR-Cas proteins interact remained unclear. To this end we purified the multi-protein RNA-guided DNA binding complex (Cascade) from the transposon-encoded minimal I-F3 CRISPR-Cas system in complex with the transposition protein TniQ. De novo modeling revealed the unexpected dimerization of TniQ, and its location within the complex, bound to the Cas6-end of the transposon-encoded Type I-F3 Cascade. Additional models obtained from DNA-bound structures of the complex demonstrate initial steps in target binding alongside novel conformations of Cascade subunits. This work reveals the mechanism by which the Tn6677 components guide integration and will enable rational engineering of these systems for further experimentation and tool development.
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Santos, Inês da Costa. "Betacellulin and Neurogenesis in the Adult Central Nervous System." Master's thesis, 2015. http://hdl.handle.net/10316/31230.
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Dissertação de mestrado em Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de CoimbraNeural stem cells (NSCs) reside in special niches in the adult brain, including subventricular zone (SVZ) of the lateral ventricle and subgranular zone (SGZ) of the dentate gyrus. Blood vessels are an important coumpound of the neurogenic niches as they secreate proteins such as betacellulin (BTC) that stimulate NSC proliferation, self-‐ renewal and differentiation. BTC is a member of the epidermal growth factor (EGF) family of ligands, and has been widely studied in many different contexts. Recently, it has been demonstrated that, in vitro, BTC can induce NSC proliferation, promote self-‐renewal and prevent spontaneous differentiation. In vivo, BTC can also promote neurogenesis. BTC is released into the neurogenic niche by endothelial cells of the microvasculature and by the choroid plexus (CP). It is thought that BTC activity in NSCs is mediated through ErbB1 and ErbB4 receptors whose activation stimulate the AKT and MEK signalling pathways. In my thesis I carried out analysis understanding the influence of BTC and other growth factors on NSCs in vitro using the neurosphere assay and measuring neurospheres number and size. I also studied the importance of AKT and MEK signalling pathways in NSCs cultivated in medium supplemented with different growth factors including BTC. I observed an increase in cell cycle arrest when inhibitors of both signalling pathways were added together in all culture conditions. Studies to better characterize the interaction between neurogenic niche and BTC were also carried out using newly developed visualisation techniques, SeeDB and CLARITY that allowed us to have a 3D perspective. In parallel, I made BTC conditional and BTC reporter contructs for generating mice using newly developed CRISPR/ Cas 9 technique. These mice will allow us to better understand parallel, I made BTC conditional and BTC reporter contructs for generating mice using newly developed CRISPR/ Cas 9 technique. These mice will allow us to better understand the relative importance of BTC in adult NSCs and whether BTC transcription is modulated
As células estaminais neuronais estão localizadas em nichos celulares especializados no cérebro adulto, incluindo a zona subventricular (ZSV) no ventrículo lateral e a zona subgranular (ZSG) no giro denteado. Os vasos sanguíneos são um componente importante dos nichos neurogenicos pois eles secretam proteínas como é o caso da betacelulina (BTC) que estimula a proliferação, auto-‐renovação, e diferenciação das células estaminais neuronais. A BTC é um membro da família de ligandos do factor de crescimento epidermal, e tem sido amplamente estudada em muitos contextos diferentes. Recentemente, foi demonstrado que, a BTC induz a proliferação, promove a auto-‐ renovação e previne a diferenciação espontânea das células estaminais neuronais, in vitro. In vivo, a BTC também promove a neurogénese. A BTC é libertada no nicho neurogénico pelas células endoteliais da microvasculatura e pelo plexo coróide. Pensa-‐se que a atividade da BTC nas células estaminais neuronais é mediada pelos receptores ErbB1 e ErbB4, cuja ativação estimula as vias de sinalização AKT e MEK. Na minha tese, procedi a análises por forma a perceber a influência da BTC e outros factores de crescimento nas células estaminais neuronais, in vitro, usando para isso o ensaio de neuroesferas bem como medindo o diâmetro e contando o número de neuroesferas. Também estudei a importância das vias de sinalização AKT e MEK nas células estaminais neuronais postas em cultura em meio suplementado com diferentes factores de crescimento incluindo a BTC. Em todas as condições de cultura, eu observei um aumento da paragem do ciclo celular quando foram adicionados os inibidores de ambas as vias de sinalização em conjunto. Foram também levados a cabo estudos para melhor perceber a interação entre o nicho neurogénico e a BTC usando para isso novas técnicas de visualização , como é o caso do SeeDB e do CLARITY que nos permitiu ter uma perspectiva 3D dessa mesma interação. Em paralelo, eu criei construções para gerar ratos condicionais e ratos repórter, usando a nova técnica CRISPR/Cas 9. Estes ratos irão permitir-‐nos melhor perceber a importância relativa da BTC nas células estaminais adultas e também perceber onde a transcrição da BTC é modulada
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Gebler, Christina. "Developing the CRISPR/Cas-system for Inactivation of Proto-oncogenes in Human Cancer Cells." Doctoral thesis, 2017. https://tud.qucosa.de/id/qucosa%3A31120.
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Numerous mutations contribute to tumorigenesis of cancer cells. For most of them it remains unclear whether they are driver or passenger mutations. A classic knock-out to study their function in cancer cells used to take a lot of effort. The CRISPR/Cas-system can be used as a programmable “genome editing” tool. In this work, oncogenes have been inactivated with the CRISPR/Cas-system.
Considering off-targets, Streptococcus pyogenes sgRNAs can be designed for 88% of the known cancer mutations. The activity of 15 sgRNAs, targeting 13 mutations in proto-oncogenes (deletions, insertions and point mutations), has been tested with a RFP-GFP-reporter plasmid. For 13 sgRNAs, activity prediction scores correlated with measured activity. Furthermore, sgRNAs have shown preferential binding to mutated versions of targeted proto-oncogene sequence and did not induce double strand breaks in the wild type sequence. For 10 sgRNAs, the activity against their target sequence has been more than 4 times higher than against the wild type sequence. Most of those sgRNAs target insertions or deletions and fewer target point mutations.
Permanent knock-out of three mutated proto-oncogenes NPM1, BRAF and PIK3CA has been achieved with a lentiviral expression of CRISPR/Cas. Accordingly, effects on proliferation and phenotype have been studied.
Knock-out of NPM1 c.863_864insTCTG mutation has been studied in heterozygous mutated OCI AML3 cell line. Proliferation was strongly inhibited by the corresponding sgRNA. Cells arrested in G0/1-phase of cell cycle (77%) compared to control cells (56%), although no difference was observed for sub-G1 phase, indicating no induction of apoptosis. Cells treated with NPM1 sgRNA had 88% reduced expression of NPM1 c.863_864insTCTG mRNA as well as less cytoplasmic localization of nucleophosmin as assessed by immunostaining. The activity of sgRNA has been confirmed by deep sequencing, showing a shift of wild type to mutated allele ratio from 51:49 to 68:32. This effect was enhanced by the additional treatment with the NHEJ inhibitor SCR7.
A BRAFV600E sgRNA was tested in homozygously mutated melanoma cell lines A-375 and SK MEL-28. No differences were detected in comparison to controls. However, in the CRC cell line RKO, heterozygous for BRAFV600E and PIK3CAH1047R, proliferation was inhibited through sgRNAs against either BRAF or PIK3CA. A combination of both had no synergistic effect on proliferation. Activity and specificity of the sgRNA targeting BRAF were confirmed by deep sequencing, while the PIK3CA sgRNA showed a moderate induction of double strand breaks also in the wild type allele. The relation of wild type to mutated allele of BRAF was changed from 32:68 before treatment to 51:49 afterwards. This effect can be explained by a “re mutation” to the wild type after DSB via HDR with wild type sister chromatid as template. This effect was observed for PIK3CA sgRNA to a lesser extent.
In conclusion, these results show the applicability of the CRISPR/Cas-system for the inactivation of mutated proto-oncogenes.:List of tables III
List of figures IV
List of abbreviations V
1 Introduction 1
1.1 Cancer 1
1.2 Oncogenes 2
1.2.1 Role in cancer 2
1.2.2 Targeted therapies 3
1.2.3 NPM1 5
1.2.4 BRAF 6
1.2.5 PIK3CA 7
1.3 CRISPR/Cas-system 7
1.4 Aim and motivation 10
2 Material and Methods 11
2.1 Design of sgRNAs 11
2.2 Plasmids 11
2.3 Cell culture 12
2.4 FACS analysis 14
2.5 T7 assay 14
2.6 Cell cycle analysis 15
2.7 Immunostaining 15
2.8 Apoptosis assay 16
2.9 Quantification of mutant NPM1 transcripts 16
2.10 Deep sequencing 16
2.11 Statistical Analysis 19
3 Results 20
3.1 Design of sgRNAs targeting oncogenes 20
3.2 Evaluation of sgRNA efficacy and selectivity 23
3.3 Effects of oncogene knock-out in cancer cell lines 27
3.3.1 Targeting NPM1 in AML cells 27
3.3.2 Targeting BRAF in melanoma cells 30
3.3.3 Targeting BRAF and PIK3CA in colorectal carcinoma cells 31
4 Discussion 37
4.1 The design of sgRNAs is possible for most cancer mutations 37
4.2 sgRNAs targeting oncogenes have to be tested 37
4.3 Oncogenes can be knocked out with the CRISPR/Cas-system 37
4.3.1 NPM1 in AML cells 37
4.3.2 BRAF in melanoma cells 38
4.3.3 BRAF and PIK3CA in CRC cells 38
4.4 Advantages and disadvantages to target oncogenes with the CRISPR/Cas-system 40
4.5 Concluding remarks 41
5 Original Article 43
6 Summary 47
7 Zusammenfassung 49
List of references 51
Appendix VIIIIn Krebszellen tritt eine Vielzahl von Mutationen auf. Für den Großteil der Mutationen ist ungeklärt, ob es sich um krebsverursachende oder passagere Mutationen handelt. Ein gezieltes Ausschalten (Knock-out) dieser Gene zur Untersuchung ihrer Funktion in Krebszellen war bisher mit großem Aufwand verbunden. Das CRISPR/Cas-System lässt sich als programmierbares „Genome-editing“ Werkzeug einsetzen und wurde in der vorliegenden Arbeit verwendet, um gezielt mutierte Protoonkogene zu inaktivieren. Für 88% der bekannten, in Krebszellen auftretenden Mutationen lassen sich, unter Berücksichtigung von off-targets, Streptococcus pyogenes sgRNAs entwerfen. Mit Hilfe eines RFP-GFP-Reporter-Plasmides wurde die Aktivität von 15 sgRNAs gegen 13 Mutationen (Deletionen, Insertionen und Punktmutationen) in Protoonkogenen überprüft. Für 13 der sgRNAs zeigte sich eine Aktivität, die mit der Vorhersage durch den Algorithmus korrelierte. Außerdem wurde gezeigt, dass die sgRNAs spezifisch genug binden, um zwar bei der mutierten Sequenz eines Protoonkogens, jedoch nicht bei der Wildtyp-Sequenz Doppelstrangbrüche zu erzeugen. Unter den sgRNAs waren 10 mit mehr als 4-fach höherer Aktivität bei komplett übereinstimmender Zielsequenz gegenüber der Wildtyp-Sequenz. Diese spezifischen sgRNAs waren vor allem gegen Insertions- oder Deletionsmutationen gerichtet, einige auch gegen Punktmutationen. Durch permanente, lentivirale Expression von CRISPR/Cas wurden die Effekte eines Knock-out von drei mutierten Protoonkogenen, NPM1, BRAF und PIK3CA, auf das Wachstum und phänotypische Aspekte humaner Krebszelllinien untersucht. Ein Knock-out der NPM1 c.863_864insTCTG Mutation wurde in heterozygot mutierten OCI AML3 Zellen untersucht, es zeigte sich eine starke Proliferationshemmung. In der Zellzyklusanalyse trat ein G0/1-Arrest dieser Zellen (77%) im Vergleich mit Kontroll-Zellen (56%) auf, jedoch keine Unterschiede in der sub-G1-Analyse, sodass nicht von einer vermehrten Apoptose auszugehen ist. Die mit sgRNA behandelten OCI-AML3 Zellen zeigten sowohl eine um 88% verminderte NPM1 c.863_864insTCTG mRNA-Expression als auch verminderte zytoplasmatische Sublokalisation des Nucleophosmins in der Immunfärbung. Die hohe Aktivität der gRNA gegen mutiertes NPM1 wurde durch Deep Sequencing bestätigt, außerdem hat sich das Verhältnis vom Wildtyp- zu mutiertem Allel von 51:49 zu 68:32 verschoben. Dieser Effekt wurde durch Zugabe des NHEJ-Hemmstoffes SCR7 noch verstärkt. Die sgRNA gegen BRAFV600E wurde in den homozygot mutierten Melanom-Zelllinien A-375 und SK-MEL-28 getestet. Bei Proliferationsversuchen zeigten sich keine Unterschiede im Vergleich zu Kontrollzellen. In der kolorektalen Krebszelllinie RKO, die heterozygot BRAFV600E und PIK3CAH1047R ist, zeigte sich bei der Testung von sgRNAs gegen BRAF, PIK3CA und Kombination beider sgRNAs eine Wachstumshemmung. Jedoch lag kein synergistischer Effekt bei sgRNA-Kombination vor. Zudem bestätigten sich Aktivität und Spezifität der sgRNA gegen BRAF im Deep Sequencing, während die sgRNA gegen PIK3CA in mäßigem Umfang Doppelstrangbrüche im Wildtyp-Allel verursachte. Das Verhältnis vom Wildtyp- zu mutiertem BRAF Allel verschob sich von 32:68 ohne sgRNA zu 51:49 nach sgRNA-Behandlung. Eine mögliche Erklärung dieser Beobachtung ist die Rückmutation zum Wildtyp-Allel nach Doppelstrangbruch mit Hilfe homologer Rekombination durch das Wildtyp-Schwesterchromatid. Für PIK3CA konnte dieser Effekt in schwächerem Ausmaß ebenfalls beobachtet werden. Zusammengefasst zeigen diese Ergebnisse, dass das CRISPR/Cas-System zur Inaktivierung mutierter Protoonkogene genutzt werden kann.:List of tables III List of figures IV List of abbreviations V 1 Introduction 1 1.1 Cancer 1 1.2 Oncogenes 2 1.2.1 Role in cancer 2 1.2.2 Targeted therapies 3 1.2.3 NPM1 5 1.2.4 BRAF 6 1.2.5 PIK3CA 7 1.3 CRISPR/Cas-system 7 1.4 Aim and motivation 10 2 Material and Methods 11 2.1 Design of sgRNAs 11 2.2 Plasmids 11 2.3 Cell culture 12 2.4 FACS analysis 14 2.5 T7 assay 14 2.6 Cell cycle analysis 15 2.7 Immunostaining 15 2.8 Apoptosis assay 16 2.9 Quantification of mutant NPM1 transcripts 16 2.10 Deep sequencing 16 2.11 Statistical Analysis 19 3 Results 20 3.1 Design of sgRNAs targeting oncogenes 20 3.2 Evaluation of sgRNA efficacy and selectivity 23 3.3 Effects of oncogene knock-out in cancer cell lines 27 3.3.1 Targeting NPM1 in AML cells 27 3.3.2 Targeting BRAF in melanoma cells 30 3.3.3 Targeting BRAF and PIK3CA in colorectal carcinoma cells 31 4 Discussion 37 4.1 The design of sgRNAs is possible for most cancer mutations 37 4.2 sgRNAs targeting oncogenes have to be tested 37 4.3 Oncogenes can be knocked out with the CRISPR/Cas-system 37 4.3.1 NPM1 in AML cells 37 4.3.2 BRAF in melanoma cells 38 4.3.3 BRAF and PIK3CA in CRC cells 38 4.4 Advantages and disadvantages to target oncogenes with the CRISPR/Cas-system 40 4.5 Concluding remarks 41 5 Original Article 43 6 Summary 47 7 Zusammenfassung 49 List of references 51 Appendix VIII
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Rajado, Ana Teresa Amado Mateus Santos. "Computation meets experimentation to improve the catatysis and specificity of Cas12a genome editing enzyme." Master's thesis, 2020. http://hdl.handle.net/10316/92173.
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Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e TecnologiaO sistema CRISPR-Cas é uma ferramenta aplicada a edição genética. Esta técnica tornou-se altamente relevante nos últimos anos devido ao seu baixo custo e facilidade de produção e utilização.Cas12a é uma endonuclease do tipo V do Sistema CRISPR-Cas, capaz de editar genoma humano recorrendo a um único RNA guia. Esta enzima já foi adaptada e utilizada em diversas áreas, tal como medicina e agricultura, através da edição genética de células de diferentes tipos, como por exemplo células animais e vegetais. No entanto, este sistema também enfrenta alguns problemas, dos quais se destacam as mutações introduzidas fora dos locais alvo (“off-target mutations”), que são introduzidas de forma não intencional.O objetivo deste trabalho é estudar o mecanismo catalítico da enzima Cas12a, com o intuito de aumentar a especificidade do mesmo. Com este propósito recorremos a uma combinação de métodos computacionais (Dinâmica Molecular) e experimentais (Biologia Molecular), para reduzir os efeitos “off-target” acima mencionados.Foram estudadas seis variantes da enzima nativa (direcionadas para as regiões da enzima que interagem com o motivo PAM, com o loop no terminal 5’ do crRNA e com o centro ativo da enzima) e dois estados intermediários do ciclo catalítico da mesma. Com as variantes criadas induzimos interações mais fortes do que as previamente presentes entre a FnCas12a, uma enzima para edição genética, e o crRNA e DNA alvo a ela associados. Substituímos resíduos polares e não polares por lisinas, carregadas positivamente, criando interações carga-carga com o DNA e o crRNA, o que poderá conduzir ao reconhecimento específico entre a proteína e os ácidos nucleicos através de um mecanismo de reconhecimento indireto (indirect readout mechanism), uma vez que os resíduos mutados não interagem com as bases azotadas.Explorámos também o mecanismo catalítico desta enzima, ao estudarmos a relevância dos resíduos H922 e R1218, localizados no local catalítico da enzima. De acordo com as nossas simulações, H922 aparenta ser o resíduo que atua como base catalítica através de um mecanismo concertado. Neste, a histidina deverá receber um protão da água enquanto que ao mesmo tempo esta realiza um ataque nucleofílico ao grupo fosfato em que irá ocorrer a clivagem.
The CRISPR-Cas system is a tool used for genome editing that became highly relevant in the latest years for being cheap, easy to design and produce.Cas12a is an endonuclease type V of the CRISPR-Cas system and is able to edit human genome through a single-RNA guided approach. This enzyme has already been repurposed to be applied in several fields, such as in medicine and agriculture, through the genome editing of different cells types such animal and plant cell. However a recurrent problem of these systems and related ones is the off-target mutations - unintentionally induced.The objective of this work is to study Cas12a enzyme. For this, we used a combination of computational (Molecular Dynamics) and experimental (Molecular Biology) methods, in order to surpass the above mentioned obstacles.Six variants of the wild type enzyme (directed to enzyme’ regions that interact with the PAM motif, the crRNA 5’ handle and with the active site of the protein) and two putative intermediates of its catalytic cycle were tested. With these variants, we induced stronger interactions between FnCas12a, a genome editing enzyme, and its crRNA and target DNA. We have substituted polar and non-polar residues with positively charged lysine residues creating new salt-bridges with the cRNA and DNA and possibly leading to specific recognition through an indirect readout mechanism, since the newly introduced residues do not interact with the bases.Additionally, we explored the catalytic mechanism of this enzyme, by studying the relevance of H922 and R1218, residues located in the catalytic site of the enzyme. According to our simulations, H922 seems to be the most probable residue to act as a base through a concerted mechanism, in which it receives a proton from the water, simultaneously with nucleophilic attack on the phosphate group that is going to be cleaved.
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Po-XingZheng and 鄭伯忻. "Relationship of CRISPR-cas system with emm typing and erythromycin susceptibility in Streptococcus pyogenes." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/y3ft35.
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博士國立成功大學
基礎醫學研究所
103
Clustered regularly interspaced short palindromic repeats (CRISPR) are the bacterial adaptive immune system against the foreign nucleic acid. Given the variable nature of CRISPR, it would be a good marker for molecular epidemiology. Streptococcus pyogenes is one of the major human pathogens. It has two CRISPR loci, including CRISPR01 and CRISPR02. The first aim of this study was to analyze the distribution of CRISPR-associated genes cassette (cas) and CRISPR arrays in highly prevalent emm types. Since antibiotic-resistant genes are frequently encoded in foreign nucleic acids, the second aim was to analyze whether erythromycin susceptibility is associated with characteristics of CRISPR elements. In the first part, the cas cassette and CRISPR array in two CRISPR loci were analyzed in a total of 332 strains, including emm1, emm3, emm4, emm12, and emm28 strains. All strains had at least one cas cassette or CRISPR array. More than 90% of the spacers were found in one emm type, specifically. Comparing the consistency between emm and CRISPR types by Simpson’s index of diversity and adjusted Wallace coefficient, CRISPR01 type was correlated with the emm type of S. pyogenes, and CRISPR02 showed unidirectional congruence to emm type, suggesting CRISPR typing can be used as an alternative way to analyze the emm type of S. pyogenes. In the second part, erythromycin susceptibility of 330 isolates collected during 1997-2003 was analyzed. Among 29 emm types, emm12, emm75, and emm92 showed significant changes in erythromycin-resistance rates. By analyzing the spacers from two CRISPR loci, I found that spacer contents in emm12, emm75, and emm92 strains were associated with erythromycin susceptibility. The strains with fewer spacers were more resistant to erythromycin. Moreover, in emm4 strains, shown no significant change in their annual erythromycin-resistance rate, CRISPR type and number of spacers were not associated with erythromycin susceptibility. These results highlight a novel association between CRISPR spacer content and erythromycin susceptibility in S. pyogenes. Together, these evidences indicate that CRISPR is a good marker for molecular epidemiology in S. pyogenes.
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Kuan, Shu-Min, and 官舒敏. "Structural study of the Cas6 protein of the CRISPR/Cas system from Methanocaldococcus jannaschii." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/35350699432300671635.
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Plagens, André [Verfasser]. "Characterisation of the CRISPR/Cas system of the hyperthermophilic Archaeum Thermoproteus tenax / vorgelegt von André Plagens." 2010. http://d-nb.info/1007172762/34.
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Yang, Chi-Dung, and 楊冀冬. "Regulatory roles of CRP on the CRISPR/Cas system, small RNAs, and lacI gene in Escherichia coli." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/udfh5a.
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博士國立交通大學
生物科技系所
102
Cyclic adenosine monophosphate (cAMP) receptor protein (CRP), activated by cAMP in low-glucose environments, is an important transcription factor in Escherichia coli that participates in the gene expression for non-glucose carbon source metabolism. Research in the past 30 years revealed that CRP controls more than 400 genes. Recent discoveries of other important genetic control systems, such as the clustered regularly interspaced short palindromic repeat/Cas system (CRISPRC/Cas system), small ribonucleic acids (sRNAs), and feed-forward loops (FFLs), demonstrated the complexity of the gene regulation network. CRP could accomplish various regulations in E. coli by regulating different genetic control systems. The CRISPRC/Cas system in bacteria could combat phage infection by producing CRISPR–RNA (crRNA). We revealed that the addition of glucose to the medium inhibited the replication capacity of phage P1 in E. coli because of the regulation of crRNA by CRP. sRNAs are important regulatory factors. sRNAs are approximately 40 bp to 500 bp in length and are located in the noncoding region. More than 80 sRNAs with various physiological functions have been reported in E. coli. More than a third of such sRNAs was regulated by CRP, forming FFLs in different forms. The coherent type 1 FFLs could maximize the acceleration of gene expression, and the incoherent type 1 FFLs delayed response to environmental messages. Thus, the formation of FFLs contributed to the adaptability of E. coli to the environment. In addition, CRP repressed lacI gene and formed CRP-LacI-lacZ FFL with lacZ (β-Galactosidase). The interdependent regulatory relationships among these three genes formed the coherent type 4 FFL. Thus, E. coli may use dual carbon (e.g., glucose and lactose) more efficiently during development under different environments.
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Hagmann, Hanns Antony. "The impact of the CRISPR/Cas system on the interaction of Neisseria meningitidis with human host cells." Doctoral thesis, 2020. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-199490.
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Neisseria meningitidis, a commensal β-proteobacterium residing exclusively in the human nasopharynx, is a leading cause of sepsis and epidemic meningitis worldwide. While comparative genome analysis was able to define hyperinvasive lineages that are responsible for most of the cases of invasive meningococcal disease (IMD), the genetic basis of their virulence remains unclear. Recent studies demonstrate that the type II C CRISPR/Cas system of meningococci is associated with carriage and less invasive lineages. CRISPR/Cas, an adaptive defence system against foreign DNA, was shown to be involved in gene regulation in Francisella novicida. This study shows that knockout strains of N. meningitidis lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells in a strain-dependant manner, which constitutes a central step in the pathogenesis of IMD. Consequently, this study indicates that the meningococcal CRISPR/Cas system fulfils functions beyond the defence of foreign DNA and is involved in the regulation of meningococcal virulenceNeisseria meningitidis, ein ß-Proteobakterium, welches als Kommensale ausschließlich den humanen Nasopharynx besiedelt, ist ein weltweit führender Verursacher von Sepsis und epidemischer Meningitis. Auch wenn mittels vergleichender Genomanalysen hyperinvasive Stämme definiert werden konnten, welche für die meisten Fälle von invasiven Meningokokkenerkrankungen verantwortlich sind, bleibt die genetische Grundlage ihrer Virulenz ungeklärt. In vorangegangenen Studien konnte gezeigt werden, dass das Typ II-C CRISPR/Cas-System der Meningokokken assoziiert ist mit Trägerstämmen. CRISPR/Cas ist ein adaptives Verteidigungssystem der Bakterien gegen fremde DNA, das darüber hinaus Aufgaben in der Genregulation von Francisella novicida erfüllt. Diese Arbeit zeigt, dass knockout Stämme von N. meningitidis, denen das Cas9-Protein fehlt, in Abhängigkeit von ihrem genetischen Hintergrund die Fähigkeit verlieren an Zellen des menschlichen Nasopharynx zu adhärieren. Die Adhäsion an den Wirtszellen stellt einen zentralen Schritt in der Pathogenese der invasiven Meningokokkenerkrankungen dar. Die Ergebnisse dieser Arbeit deuten darauf hin, dass das CRISPR/Cas-System in Meningokokken neben seiner Funktion als bakterielles Immunsystem an der Regulation der bakteriellen Virulenz beteiligt sein könnte