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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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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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Roidos, Paris. "Genome editing with the CRISPR Cas9 system." Thesis, KTH, Skolan för bioteknologi (BIO), 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-163694.
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Ellis, Donald Christian. "Genetic screens in vivo using the CRISPR/Cas9 system." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/109640.
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Thesis: S.M., Massachusetts Institute of Technology, Department of Biology, 2017.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 82-87).
An unmet and paramount need in the field of cancer research is to rapidly translate basic biological findings to clinically relevant therapeutics for cancer patients. Recent technological advances have generated many innovative applications to cancer biology and in a short time have yielded a wealth of information about putative vulnerabilities across a range of cancers. The proposed work involves the development of a technique to quickly probe potential cancer-specific vulnerabilities in vivo adopting methods used in genetic screens. By harnessing the information obtained from large datasets in vitro and the utility of cutting-edge endogenous mouse models, the general aim of this work is to create a method that shortens the gap between findings in the lab to viable treatment options for cancer patients.
by Donald Christian Ellis.
S.M.
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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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Arooj, Mahira. "Precision Genome Engineering and Gene Silencing Using CRISPR/dCas9 System." Thesis, Curtin University, 2018. http://hdl.handle.net/20.500.11937/73571.
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This research aimed to repress target genes by modulating their expression through rewriting of aberrant epigenetic landscapes, including DNA methylation and histone post-translational modifications, utilizing epigenetic repressor domains. The epigenetic repressor domains included KRAB, DNMT3A, UHRF1 and CSD, which were delivered via the epigenetic editing tools ZFs, TALEs and the CRISPR/dCas9 system. Our findings suggest that a combination of multiple epigenetic repressor domains targeting epigenetic marks involved in epigenetic cross-talk significantly repressed target genes.
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Dhanjal, Jaspreet Kaur. "Computational insights into CRISPR/Cas9 system for improved genome editing." Thesis, IIT Delhi, 2019. http://eprint.iitd.ac.in:80//handle/2074/8077.
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Cui, Xiucheng. "Targeted Gene Editing Using CRISPR/Cas9 in a Wheat Protoplast System." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/36543.
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The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has become a promising tool for targeted gene editing in a variety of organisms including plants. In this system, a 20 nt sequence on a single guide RNA (sgRNA) is the only gene-specific information required to modify a target gene. Fusarium head blight (FHB) is a devastating disease in wheat caused by the fungus Fusarium graminearum. The trichothecene it produces, deoxynivalenol (DON), is a major mycotoxin contaminant causing food production loss both in quality and yield. In this project, we used the CRISPR/Cas9 system to modify three wheat genes identified in previous experiments, including an ABC transporter (TaABCC6), and the Nuclear Transcription Factor X box-binding-Like 1 (TaNFXL1), both associated with FHB susceptibility, and a non-specific Lipid Transfer Protein (nsLTP) named TansLTP9.4 which correlates with FHB resistance. Two sgRNAs were designed to target each gene and were shown in an in vitro CRISPR/Cas9 assay to guide the sequence-specific cleavage with high efficiency. Another assay for CRISPR/Cas9 was established by the optimization of a wheat protoplast isolation and transformation system. Using a construct expressing a green fluorescent protein (GFP) as a positive control, estimated transformation efficiencies of about 60% were obtained with different batches of protoplasts. High-throughput sequencing of PCR amplicons from protoplasts transformed with editing constructs clearly showed that the three genes have been successfully edited with efficiencies of up to 42.2%. In addition, we also characterized by RT-qPCR the expression pattern of 10 genes in DON-treated protoplasts; seven of the genes were induced by DON in the protoplasts, consistent with their previously identified DON induction in treated wheat heads, while three genes expressed differentially between DON-treated wheat heads and protoplasts. Preliminary bioinformatics analyses showed that these differentially expressed genes are involved in different plant defense mechanisms.
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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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Seo, Jooheon. "Modulation of DNA repair pathway after CRISPR/Cas9 mediated Double Stranded Break." Thesis, Virginia Tech, 2017. http://hdl.handle.net/10919/74884.
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The CRISPR/Cas9 system has become the predominant tool for genome editing. Targeted modifications can be introduced while repairing double strand breaks (DSBs), induced by the CRISPR/Cas9 system. The DSB is repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR), and the repair is commonly processed through NHEJ because it is the dominant repair pathway in most cell types. The goal of this study is to modulate DNA repair system of somatic cells to increase the frequency of homology-directed repair (HDR) through HR by chemical treatment and the frequency of NHEJ by serum starvation. CRISPR/Cas9 systems targeting RAG2 gene and donor DNA to replace endogenous RAG2 were transfected into porcine fetal fibroblast (PFF) cells and the cells were treated with various chemicals that were known to inhibit NHEJ or stimulate HR. Among the chemical treated groups, cells treated with thymidine showed an average of 5.85-fold increase in HDR compared to the control group; the difference ranged from 1.37 to 9.59. There was no positive effect on the frequency of HDR after treating transfected cells with other chemicals. Placing PFFs under low amount of serum (serum deprivation) could enrich the cells in G0/G1 phase, but there was little difference in the frequency of NHEJ. Our results indicate that modulating DNA repair pathways during CRISPR/Cas9-mediated gene targeting could change the outcome of the targeted events.Master of Science
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Smargon, Aaron Andrew. "Discovery and characterization of Cas13b, a differentially regulated RNA-targeting CRISPR system." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/115637.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2018.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 129-144).
RNA plays a significant role in human biology and disease, not only as messenger RNA encoding proteins but also as noncoding RNA regulating DNA, proteins, and other RNA species. Until recently, it has been challenging to target RNA in a simple, efficient manner. CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) systems, which confer adaptive immunity to prokaryotes, have revolutionized DNA targeting through the engineering of RNA-programmable Cas9-based tools. Effective RNA-programmable RNA-targeting tools would likewise transform RNA biology and biotechnology. Class 2 CRISPR-Cas systems, which rely only on a single effector protein and programmable CRISPR RNA (crRNA) to target nucleic acids, represent the most promising tool to target RNA. Building on previous research, a biocomputational pipeline was developed to discover novel functional class 2 CRISPR systems lacking the canonical adaptive machinery of Cas1 and Cas2 at their genomic loci. Out of this pipeline emerged the class 2 CRISPR-Cas RNA-targeting system, VI-B (Cas13b with accessory Csx27/Csx28). Cas13b was characterized both biochemically and genetically, and found to be differentially regulated--inhibited by Csx27 in VI-B1 systems and enhanced by Csx28 in VI-B2 systems. RNA-targeting rules are critical to tool development, and so an E. coli essential gene screen was conducted and analyzed to assess the RNA sequence and structure requirements for targeting. The completion of this work advances both knowledge in the CRISPR field and possibilities in the RNA-targeting toolkit.
by Aaron Andrew Smargon.
Ph. D.
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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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O'Sullivan, Harrison. "CRIPTIC-12a: A novel CRISPR Programmable Transposon Insertion system using Cas12a (Cpf1)." Thesis, O'Sullivan, Harrison (2020) CRIPTIC-12a: A novel CRISPR Programmable Transposon Insertion system using Cas12a (Cpf1). Honours thesis, Murdoch University, 2020. https://researchrepository.murdoch.edu.au/id/eprint/63541/.
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The Acidaminococcus sp. enzyme AsCpf1 has recently been adopted for gene editing purposes since it can be guided to a T-rich PAM recognition site instead of the G-rich PAM recognition site of the widely used SpCas9. Both systems engineer targeted double stranded breaks in DNA that can be repaired by non-homologous end-joining (NHEJ) or homology directed repair (HDR) mechanisms to generate genetic mutations in eukaryotes.
In prokaryotes that lack NHEJ and HDR repair processes, random mutation can be introduced via transposase enzymes that cleave dsDNA and insert transposable elements into these locations. The TnpATn5 protein, native to the prokaryote Escherichia coli, binds to a set of inverted terminal repeats that flank the transposon Tn5, or its derivatives, and excises the element before randomly inserting this transposable element into a gene to create a knock-out mutation.
In this thesis a novel strategy was designed to fuse a catalytically inactive AsdCpf1 endonuclease to TnpATn5-NB/HYP (a modified Escherichia coli Tn5 transposase) to produce the chimeric protein AsdCpf1-TnpATn5-NB/HYP that could be used to produce programmable transposition. Domain and 3D protein analysis were used to theorize the best structural configuration of the fusion between AsdCpf1 and TnpATn5-NB/HYP. The TnpATn5-NB/HYP component was engineered to contain six mutations; four of which were used to make it non-biased, and two of which were used to make the transposase hyperactive.
To ascertain whether the fused protein components were functional, two highly regulated modular bioassays were developed and implemented. A tripartite plasmid bioassay was used to detect repression by AsdCpf1 or AsdCpf1-TnpATn5-NB/HYP, and a quadripartite plasmid bioassay was used to detect non-targeted and targeted insertion events catalyzed by AsdCpf1-TnpATn5-NB/HYP.
The core plasmid system was tripartite and contained compatible oriV elements and distinct antibiotic resistance markers to force for plasmid retention. The first plasmid was engineered to carry the programmable guide production module gCPF1 and the AsdCpf1 or AsdCpf1-TnpATn5-NB/HYP effector modules. The “CHOPCHOP” algorithm predicted guides were cloned into the gCPF1 module at directional BsaI sites. The second plasmid contained the lacIq regulatory component, and either the lacZα (colourimetric detection) or sacB (viability assay) genes required for the repression bioassays. The third plasmid contained a LacIq regulated T7p07 gene, the product of which transcribed the crRNA in the gCPF1 module. This system could be switched from an OFF state to an ON state in the absence or presence of IPTG, respectively. In the ON state, the single copy lacZω sequence on the E. coli chromosome was noticeably repressed by two of the three guides complexed with AsdCpf1. The sacB sequence introduced on a multi-copy plasmid could only be noticeably repressed by one of the guides complexed with AsdCpf1. This thesis showed repression was highly dependent on both guide sequence location and gene copy number. The assay repeated with the chimeric protein revealed that repression was occurring, albeit at a diminished level.
The core AsdCpf1-TnpATn5-NB/HYP system was expanded to a quadripartite bioassay that included a suicidal R6K plasmid delivering a mTn5 transposable element. Using this system in the OFF state, transposition was shown to occur into random sites that excluded lacZω. In contrast, using this system in the ON state, transposition only occurred in the lacZω gene, revealing functional guided transposition could be obtained. The successful creation of the chimeric AsdCpf1-TnpATn5-NB/HYP protein in this thesis provides an essential foundation for further work developing Cas-Tnp fusion proteins for work in prokaryotic hosts. It also provides another step towards large-scale gene silencing and editing with extraordinary accuracy.
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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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Gergen, Janina. "The CRISPR/Cas9 system as an anti-viral strategy against the human cytomegalovirus." Thesis, Nantes, 2017. http://www.theses.fr/2017NANT1036/document.
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L’infection primaire par le cytomégalovirus (CMV) humain est asymptomatique. Le sujet infecté reste cependant porteur du virus à l’état latent. Le CMV ne se réactive que sporadiquement chez l’individu immunocompétent. Chez les patients immunodéprimés, la réactivation du CMV peut induire des maladies à CMV touchant des organes vitaux et peut mettre en jeu le pronostic vital du patient. Les traitements standards sont efficaces mais leurs effets secondaires et l’apparition de souches virales résistantes relancent l’engouement pour le développement de nouvelles thérapies. Lors de ma thèse, j’ai utilisé le système CRISPR/Cas9 afin de déstabiliser le génome du CMV. Nous avons choisi de cibler le gène UL122/UL123 codant pour les molécules immediate early essentielles au cycle lytique réplicatif et à la sortie de la latence. Nous avons comparé deux stratégies utilisant soit un soit trois gRNAs, respectivement appelées singleplex et multiplex ciblant ce même gène. Alors que le singleplex induit des insertions et délétions au site de coupure du gRNA, la stratégie multiplex induit la délétion de 3500 paires de bases du gène ciblé. De ce fait, la stratégie multiplex bloque efficacement l’expression du gène ciblé, la réplication virale et le relargage de nouveaux virions. Une autre stratégie a été développée pour cibler spécifiquement le génome à l’état latent. Les deux régions homologues TR et le gène LUNA sont ciblés par deux gRNAs. Cette seconde stratégie résulte elle aussi en une diminution du nombre de copies de génome viral lors du cycle lytique de réplication. Il est désormais possible d’envisager de nouvelles solutions thérapeutiques anti-HCMV avec la stratégie CRISPR/Cas9The human cytomegalovirus (HCMV) primary infection is usually asymptomatic but leads to latent infection of blood progenitor cells. Immunocompromised patients are at high risks of HCMV reactivation, which is associated with severe end organ diseases and increased mortality in transplant patients. Standard anti-viral treatments based on nucleotide analogues decreased the occurrence of HCMV reactivation and diseases, but induce side effects and drug-resistant viral strains. In this thesis, we introduced new anti-viral approaches based on the CRISPR/Cas9 gene editing tool. Two strategies are designed to target the UL122/123 gene of HCMV encoding the immediate early proteins, essential for lytic viral replication and reactivation from latency. We validated that the disruption of the UL122/123 gene by the CRISPR/Cas9 system to abrogate viral replication. The multiplex CRISPR/Cas9 system (three gRNA) was much more efficient than the singleplex approach targeting the same gene. Target gene expression, concomitant genome replication and virion release were significantly impaired by the multiplex strategy. A further anti-HCMV CRISPR/Cas9 system was developed specifically to target the HCMV genome during latency. Two gRNAs target the viral genome at three target sites: LUNA, essential for reactivation, and the two homolog TR regions. We verified this duplex strategy on the lytic replicating virus and detected mutations at the target site as well as the reduction of viral genome copy number. In conclusion, the anti-HCMV strategies based on two or three gRNAs efficiently blocked viral replication. This provides the basis for the development of an anti-HCMV CRISPR/Cas9 therapy
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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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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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Amai, Takamitsu. "Development of genome editing technology of mitochondrial DNA in Saccharomyces cerevisiae." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263707.
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Hirosawa, Moe. "Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch." Kyoto University, 2019. http://hdl.handle.net/2433/242421.
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Mosqueira, Diogo. "Disease modeling hypertrophic cardiomyopathy using CRISPR/Cas9 genome editing technology in human pluripotent stem cell-derived cardiomyocytes." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/51359/.
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Hypertrophic cardiomyopathy (HCM) is a prevalent genetic cardiovascular disease affecting 1:500 individuals whose cardiac function is deteriorated due to thickening of the left ventricle of the heart, mostly owing to mutations in sarcomeric genes. Modeling HCM in vitro using human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) offers promise to further investigate the disease mechanisms, towards the development of effective drugs. Herein, nickase CRISPR/Cas9 genome editing technology was harnessed to introduce the R453C pathological mutation in the MYH7 sarcomeric gene, in three healthy hPSC lines. Monoclonal hPSC lines generated displayed the mutation in one or both alleles, as confirmed by PCR-genotyping and Sanger sequencing. A monolayer cardiac differentiation protocol was applied to the generated hPSC lines, resulting in >90% cardiomyocyte purities, and expression of mutant allele(s) of the MYH7 gene was analysed by RT-PCR. High-content imaging analysis showed that mutant hPSC-CMs displayed higher expression of hypertrophic marker Brain Natriuretic Peptide (BNP), in comparison to isogenic controls. BNP expression was maximised by treatment with hypertrophic inducer Endothelin-1 and rescued by its antagonist Bosentan. Flow cytometry analysis revealed a mild increase in cell volume of mutant cardiomyocytes relative to their wild-type controls. Functional evaluation of gene-edited lines exposed higher mitochondrial respiration rates relative to the isogenic controls, with the same mitochondrial content, resulting in a trend towards oxidative stress. Further genome engineering to incorporate a calcium indicator in R453C-MYH7 lines enabled confocal line analysis of calcium transients. MYH7-mutant hPSC-CMs exhibited higher frequency of irregular events in comparison to the healthy control, faster calcium kinetics, and higher resting cytosolic calcium concentration. Integration of hPSC-CMs in Engineered Heart Tissues (EHTs) and subsequent analysis of contractile force showed that mutant lines had a hypo-contractile and negative clinotropic phenotype relative to their isogenic controls. Moreover, R453C-MYH7 hEHTs showed a more pronounced negative force-frequency relationship in comparison with the healthy lines. These phenotypes were not rescued by treatment with cardiac myosin activator Omecamtiv Mecarbil, suggesting that targeting other mechanisms indirectly related with the contractile apparatus may be a preferred route to attenuate the observed pathological changes. Finally, transcriptomic analysis of gene-edited lines showed up-regulation of genes associated with fetal gene program, hypertrophy, fibrosis, apoptosis and autophagy, indicating potential molecular mechanisms associated with the observed phenotypes and HCM progression. Overall, hPSC-CMs bearing the R453C-MYH7 mutation exhibit the main molecular and functional hallmarks of HCM, providing a physiologically-relevant platform that enables further dissection of disease mechanisms and promotes pharmacological intervention.
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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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Ryu, Junghyun. "The direct injection of CRISPR/Cas9 system into porcine zygotes for genetically modified pig production." Diss., Virginia Tech, 2019. http://hdl.handle.net/10919/101763.
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The pig has similar features to the human in aspects such as physiology, immunology, and organ size. Because of these similarities, genetically modified pigs have been generated for xenotransplantation. Also, when using the pig as a model for human diseases (e.g. cystic fibrosis transmembrane conductance regulator), the pig exhibited similar symptoms to those that human patients present. The main goal of this work was to examine the efficacy of direct injection of the CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/ CRISPR associated protein 9) in pigs and to overcome shortcomings that resulted after direct injection into the cytoplasm of developing zygotes. By using direct injection of CRISPR/Cas9 into developing zygotes, we successfully generated fetuses and piglets containing 9 different mutations. The total number of aborted fetuses was 20 and of live piglets was 55. Moreover, one issue that was encountered during the production of mutated pigs was that insertion or deletion (indel) mutations did not always introduce a premature stop codon because it did not interfere with the codon read. As a result of these triplet indel(s) mutations, a hypomorphic phenotype was presented; consequently, the mutated gene was partially functional. To prevent this hypomorphic phenotype, we introduced two sgRNAs to generate an intended deletion that would remove a DNA fragment on the genome by causing two double-strand breaks (DSB) during non-homologous end joining (NHEJ). The injection of two sgRNAs successfully generated the intended deletion on the targeted genes in embryos and live piglets. Results after using intended deletions, in IL2RG mutation pigs, did not show hypomorphic phenotypes even when a premature stop codon was not present. After using the intended deletion approach, function of the targeted genes was completely disrupted regardless of the presence or absence of a premature stop codon. Our next aim was to introduce (i.e. knock-in) a portion of exogenous (donor) DNA sequence into a specific locus by utilizing the homology direct repair (HDR) pathway. Because of the cytotoxicity of the linear form of the donor DNA, the concentration of the injected donor DNA was adjusted. After concentration optimization, four different donor DNA fragments targeting four different genes were injected into zygotes. Efficiency of knock-in was an average of 35%. Another donor DNA was used in this study which is IL2RG-IA donor DNA carried 3kb of exogenous cassette. It showed 15.6% of knock-in efficiency. IL2RG-IA Donor DNA injected embryos were transferred into surrogates, and a total of 7 pigs were born from one surrogate, but none of the 7 were positive for the knock-in. Future experiments need to be developed to optimize this approach. Overall, the direct injection of CRISPR/Cas9 is advantageous in cost, time, and efficiency for large animal production and for biomedical research. However, there are still unsolved challenges (off-targeting effects, low efficiency of knock-in, and monoallelic target mutation) that need to be elucidated for future application in humans and other species.Doctor of Philosophy
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Matsunaga, Taichi. "Single-step generation of gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9." Kyoto University, 2014. http://hdl.handle.net/2433/188689.
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McClain, Megan. "Characterization of Putative Cyclic di-GMP Binding Proteins in Streptomyces scabies using the CRISPR-Cas9 Editing System & Bioinformatics." Otterbein University Distinction Theses / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=otbndist1620461478136619.
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Djermoun, Sarah. "Le plasmide RP4 : de son utilisation comme outil antibactérien à l’étude de sa dynamique de transfert au sein de biofilm bactérien." Electronic Thesis or Diss., Lyon 1, 2023. http://www.theses.fr/2023LYO10080.
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L’étude de la dynamique de conjugaison des plasmides conjugatifs chez les bactéries Gram-négatif est la thématique centrale de recherche de notre laboratoire et autour de laquelle s’est articulé mon projet de thèse. Mes travaux de recherche ont eu pour but d’apporter de véritables connaissances sur l’étendue et l’impact de la conjugaison dans les communautés bactériennes. Le biofilm est largement considéré par la communauté scientifique comme un hotspot favorisant le transfert de gènes principalement en raison des contacts cellulaires propices qui existent dans sa structure. Or, les seules études qui ont essayé de démontrer expérimentalement que le biofilm permet d’augmenter les transferts par conjugaison n’apportent aucunes données claires sur la dynamique de ces transferts qui ont lieu dans le biofilm et comment celui-ci impacte ces transferts. L’approche que nous avons utilisée pour étudier la dynamique de conjugaison dans le biofilm repose sur un projet collaboratif entre notre laboratoire et celui du Dr Knut Drescher, basé au Biozentrum de Bâle en Suisse. Cette collaboration a permis de déployer des techniques de microscopie à fluorescence innovantes développées par nos deux laboratoires et jusqu’ici jamais utilisées dans le contexte de l’étude de la conjugaison dans le biofilm.Nous nous sommes centrés sur le plasmide RP4 qui est un plasmide conjugatif de type IncP. Retrouvé au sein de nombreux environnements naturels, il a été le modèle plasmidique principal des études qui se sont intéressées à la conjugaison dans le biofilm, et il a été largement exploité comme outil génétique par la communauté scientifique. Malgré le fait qu’il ait été très utilisé, les mécanismes de transferts du plasmide RP4 sont très peu décrits. Le plasmide RP4 s’est donc révélé comme un modèle d’étude de la conjugaison très pertinent que nous avons utilisé à la fois dans un aspect biotechnologique pour élargir le spectre d’hôte des systèmes antibactériens TAPs et à la fois dans un aspect fondamental pour étudier sa dynamique de conjugaison, que ce soit au sein d’une population E. coli cultivées en 2D et au sein d’une population E. coli structurées en biofilm 3D.Durant mes travaux de thèse, j’ai donc exploité le plasmide RP4 pour véhiculer des systèmes CRISPR antibactériens chez diverses espèces bactériennes phylogénétiquement éloignées. J’ai apporté les premières images en temps réel du transfert du plasmide RP4 en 2D et de nouvelles données très intéressantes sur la chronologie de conversion de l’ADN en double brin dans la receveuse. Enfin, une approche totalement innovante a permis d’étudier la dynamique de conjugaison du plasmide RP4 dans le biofilm. Ces résultats constituent finalement la première étude qui décrit réellement comment la conjugaison a lieu dans le biofilm et qui va au-delà en termes de compréhension sur cette dynamique grâce à l’approche 2D que nous avions mis en place. Nous démontrons que biofilm n’est pas un hotspot pour le transfert du plasmide RP4 et que les facteurs de la matrice EPS qui compose sa structure n’empêchent pas la dissémination du plasmide. Mais qu’il s’agit plutôt du stade de développement du biofilm qui va rendre possible l’accessibilité des donneurs à l’attachement aux zones de contact avec la surface, à proximité des cellules receveusesThe study of conjugation dynamics of conjugative plasmids in Gram-negative bacteria is the central research theme of our laboratory and around which my thesis project was built. The aim of my research was to provide real knowledge on the extent and impact of conjugation in bacterial communities. The biofilm is widely considered by the scientific community as a hotspot for gene transfer mainly because of the favorable cell contacts that exist in its structure. However, the only studies that have attempted to demonstrate experimentally that biofilms increase gene transfer by conjugation do not provide clear data on the dynamics of these transfers that take place in the biofilm and how the biofilm impacts these transfers. The approach we used to study the dynamics of conjugation in biofilm is based on a collaborative project between our laboratory and that of Dr. Knut Drescher, based at the Biozentrum in Basel, Switzerland. This collaboration allowed us to deploy innovative fluorescence microscopy techniques developed by our two laboratories and never used before in the context of the study of conjugation in biofilm.We focused on the RP4 plasmid which is an IncP conjugative plasmid. Found within many natural environments, it has been the primary plasmid model for studies that have focused on conjugation in the biofilm, and has been widely exploited as a genetic tool by the scientific community. Despite the fact that it has been widely used, the transfer mechanisms of the RP4 plasmid are very poorly described. The RP4 plasmid has thus proven to be a very relevant model for studying conjugation that we have used both in a biotechnological aspect to broaden the host spectrum of antibacterial TAPs systems and in a fundamental aspect to study its conjugation dynamics, both within a 2D cultured E. coli population and within a 3D biofilm structured E. coli population.During my thesis work, I therefore exploited the RP4 plasmid to carry antibacterial CRISPR systems in various phylogenetically distant bacterial species. I provided the first real-time images of the RP4 plasmid transfer in 2D and very interesting new data on the timing of DNA double-strand conversion in the recipient. Finally, a totally innovative approach allowed to study the conjugation dynamics of the RP4 plasmid in the biofilm. These results finally constitute the first study that really describes how conjugation takes place in the biofilm and that goes beyond in terms of understanding this dynamic thanks to the 2D approach that we had set up. We demonstrate that biofilm is not a hotspot for the transfer of the RP4 plasmid and that the factors of the EPS matrix that compose its structure do not prevent the dissemination of the plasmid. Rather, it is the stage of biofilm development that makes it possible for the donors to attach to the surface contact areas near the recipient cells
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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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Murakami, Yu. "Establishment of a practical gene knock-in system and its application in medaka." Kyoto University, 2020. http://hdl.handle.net/2433/253339.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第22503号
農博第2407号
新制||農||1077(附属図書館)
学位論文||R2||N5283(農学部図書室)
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 佐藤 健司, 教授 澤山 茂樹, 准教授 豊原 治彦
学位規則第4条第1項該当
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Mosaku, Olukunbi Eniola. "The use of the CRISPR-Cas9 system and iPSC-derived neurons with a SNCA mutation to model neurodegeneration." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10062570/.
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Parkinson's disease (PD) is characterised by the selective loss of dopaminergic neurons of the substantia nigra pars compacta. Patients suffer from a progressive motor disorder, defined by the presence of rigidity, resting tremor and bradykinesia. Current treatment options, relieve symptoms for a limited period, but are not curative, as the underlying molecular causes of neurodegeneration are unknown. Several causative PD mutations have been identified and could provide insight into the defective molecular pathways in PD. Multiplication or missense mutation of the SNCA gene leads to autosomal dominant PD. Alpha-synuclein, encoded by the SNCA gene, is a defining component of proteinaceous deposits found in surviving neurons in PD and a central protein in PD aetiology. Induced pluripotent stem cells (iPSCs) self-renew indefinitely and generate all germ layer lineages. Human iPSCs derived from an individual with a genetic variant known to cause disease, provide a platform to investigate the molecular basis of disease. However, genetic variation between iPSC lines can lead to functional disparities, masking or accentuating disease-specific phenotypes. Genome engineering facilitates the generation of iPSCs which differ exclusively at the locus of interest, providing a genetically stable cellular model. iPSCs from an individual with a SNCA missense mutation, G51D, and an unaffected relative were characterised, demonstrating ex vivo pluripotency was established and dopaminergic neurons could be derived. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system was exploited to introduce the G51D mutation into control iPSCs. Changes in dopamine turnover and protein metabolism were detected after differentiation of CRISPR-Cas9 generated iPSCs, now harbouring the heterozygote G51D mutation. A CRISPR-Cas9 G51D homozygote iPSC clone was generated and a reduction in the number of dopaminergic neurons produced observed. This study demonstrates human iPSCs can be used to detect phenotypic differences in specialised cells, despite the latency of PD, and before overt neurodegeneration.
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Sayed, Shady [Verfasser], Frank [Gutachter] Buchholz, and Dirk [Gutachter] Lindemann. "Identification and inactivation of cancer driver mutations using the CRISPR-Cas9 system / Shady Sayed ; Gutachter: Frank Buchholz, Dirk Lindemann." Dresden : Technische Universität Dresden, 2021. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa2-760405.
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Bella, R. "GENE EDITING TECHNOLOGIES BASED ON CRISPR-CAS9 SYSTEM FOR THE TREATMENT OF HIV: STUDIES IN VITRO AND IN VIVO." Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/543298.
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ABSTRACT
Retroviruses include two subfamilies, orthoretrovirinae and spumaretrovirinae. The human immunodeficiency virus 1 (HIV-1) belongs to the orthoretrovirinae subfamily and is the causative agent of acquired immunodeficiency syndrome (AIDS). HIV-1 infects 36.9 million people and 2.6 million children throughout the world. During primary infection HIV converts its RNA genome into DNA, which integrates into the host genome. The cellular environment present at the site of the integration may influence viral transcriptional activity. The sequestration of host transcription factors, the presence of repressor of transcription and nucleosomes and epigenetic modifications on the HIV promoter, or transcriptional modification of Tat are all conditions that influence the formation of long term viral reservoirs. The use of antiretroviral drugs has been proposed as a functional cure to control the viral load but lacks the ability to obtain viral sterilization since antiretroviral drugs can not remove the virus from latently infected cells and anatomical sanctuaries such as brain and the gut associated lymphoid tissue. In recent years gene editing strategies have been largely employed for the treatment of HIV-1. In this present study, we aimed to discover an innovative CRISPR technology specific against the HIV viral genome that can target latently infected cells and be delivered in all tissues. Initially, we performed in vitro analysis, where TZMB-1 cells containing the luciferase gene under the control of LTR were transfected with pCMV-Tat and three plasmids harboring Cas9 under the control of different regions of LTR promoter to evaluate by western blot analysis the minimal LTR promoter region able to activate Cas9 in presence of Tat. TZMB-1 cells were transduced with the lentiviruses, harboring Cas9 or gRNAs specific for the promoter region, and infected with HIV-1 to test, by PCR and luciferase assay, the presence of gene editing. Then PCR and flow cytometric analyses were performed on 2D10 cells, HIV-1 latently infected cells, to test the ability of Tat-induced Cas9 to excise viral DNA. Subsequently, was evaluated the ability of Cas9, in presence of gRNAs, to protect Jurkat cells from viral reinfection by eliminating the virus during the early stages of infection. The second part of our study was performed to test Cas9 and gRNAs specific for HIV-1 LTR and Gag regions in vivo using adeno-associated virus (AAV) as the delivery system. Tissues of HIV-1 transgenic mice and rats and humanized mice were provided by collaborators for evaluation by analyzing DNA and RNA for the presence of viral editing. Results from in vitro experiments showed the ability of Tat to activate the minimal promoter LTR, inducing gene editing in TZMb-I and 2D10 cells. The presence of Cas9 in Jurkat cells induces a reduction of viral RNA of 96% at five days from infection. Studies in vivo showed the presence of viral excision in blood, heart, liver, lung, kidney, spleen and brain in transgenic mice and a reduction of viral RNA in the blood of transgenic rats. Excision of HIV-1 was reported in the spleen, gut associated lymphoid tissue, liver, kidney, lung and brain of humanized mice with complete viral sterilization in 29% of the infected animals that were subjected to antiretroviral treatment. The absence of off-target effects was confirmed by deep sequencing analysis. Together, these data show the ability to create a Cas9-inducible system generating negative feedback against the virus while avoiding persistent Cas9 expression in the cells. The use of AAV vectors in vivo showed high delivery efficiency in the different tissues, obtaining viral sterilization for the first-time. Further experiments on humanized mice and SIV infected monkey models will show this approach combined with ART therapy may have important application for HIV-1 sterilization in clinical trials.
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Foster, Robert Graham. "Development of a modular in vivo reporter system for CRISPR-mediated genome editing and its therapeutic applications for rare genetic respiratory diseases." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/33040.
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Rare diseases, when considered as a whole, affect up to 7% of the population, which would represent 3.5 million individuals in the United Kingdom alone. However, while 'personalised medicine' is now yielding remarkable results using recent sequencing technologies in terms of diagnosing genetic conditions, we have made much less headway in translating this patient information into therapies and effective treatments. Even with recent calls for greater research into personalised treatments for those affected by a rare disease, progress in this area is still severely lacking, in part due to the astronomical cost and time involved in bringing treatments to the clinic. Gene correction using the recently-described genome editing technology CRISPR/Cas9, which allows precise editing of DNA, offers an exciting new avenue of treatment, if not cure, for rare diseases; up to 80% of which have a genetic component. This system allows the researcher to target any locus in the genome for cleavage with a short guide-RNA, as long as it precedes a highly ubiquitous NGG sequence motif. If a repair sequence is then also provided, such as a wild-type copy of the mutated gene, it can be incorporated by homology-directed repair (HDR), leading to gene correction. As both guide-RNA and repair template are easily generated, whilst the machinery for editing and delivery remain the same, this system could usher in the era of 'personalised medicine' and offer hope to those with rare genetic diseases. However, currently it is difficult to test the efficacy of CRISPR/Cas9 for gene correction, especially in vivo. Therefore, in my PhD I have developed a novel fluorescent reporter system which provides a rapid, visual read-out of both non-homologous end joining (NHEJ) and homology-directed repair (HDR) driven by CRISPR/Cas9. This system is built upon a cassette which is stably and heterozygously integrated into a ubiquitously expressed locus in the mouse genome. This cassette contains a strong hybrid promoter driving expression of membrane-tagged tdTomato, followed by a strong stop sequence, and then membrane-tagged EGFP. Unedited, this system drives strong expression of membrane-tdTomato in all cell types in the embryo and adult mouse. However, following the addition of CRISPR/Cas9 components, and upon cleavage, the tdTomato is rapidly excised, resulting via NHEJ either in cells without fluorescence (due to imperfect deletions) or with membrane-EGFP. If a repair template containing nuclear tagged-EGFP is also supplied, the editing machinery may then use the precise HDR pathway, which results in a rapid transition from membrane-tdTomato to nuclear- EGFP. Thereby this system allows the kinetics of editing to be visualised in real time and allows simple scoring of the proportion of cells which have been edited by NHEJ or corrected by HDR. It therefore provides a simple, fast and scalable manner to optimise reagents and protocols for gene correction by CRISPR/Cas9, especially compared to sequencing approaches, and will prove broadly useful to many researchers in the field. Further to this, I have shown that methods which lead to gene correction in our reporter system are also able to partially repair mutations found in the disease-causing gene, Zmynd10; which is implicated in the respiratory disorder primary ciliary dyskinesia (PCD), for which there is no effective treatment. PCD is an autosomal-recessive rare disorder affecting motile cilia (MIM:244400), which results in impaired mucociliary clearance leading to neonatal respiratory distress and recurrent airway infections, often progressing to lung failure. Clinically, PCD is a chronic airway disease, similar to CF, with progressive deterioration of lung function and lower airway bacterial colonization. However, unlike CF which is monogenic, over 40 genes are known to cause PCD. The high genetic heterogeneity of this rare disease makes it well suited to such a genome editing strategy, which can be tailored for the correction of any mutated locus.
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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.
Full textAbstract:
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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Adaui, Vanessa, Constanze Kröber-Boncardo, Christine Brinker, Henner Zirpel, Julie Sellau, Jorge Arévalo, Jean Claude Dujardin, and Joachim Clos. "Application of crispr/cas9-based reverse genetics in leishmania braziliensis: Conserved roles for hsp100 and hsp23." MDPI AG, 2020. http://hdl.handle.net/10757/655510.
Full textAbstract:
The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23-and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. major HSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR–Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite’s biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.Alexander von Humboldt-Stiftung
Revisión por pares
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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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Sindi, Abdulmajeed Abdulghani A. "Investigating the role of HMGN2 in the self-renewal and neuronal differentiation of ECCs using the CRISPR-Cas9 knockout system." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8591/.
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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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Kapahnke, Marcel [Verfasser]. "Knock-out of Flotillins in Human Cells Using the CRISPR-Cas9 Genome Editing System: Effects on mRNA Splicing / Marcel Kapahnke." Gießen : Universitätsbibliothek, 2020. http://d-nb.info/1223462137/34.
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Satomura, Atsushi. "Studies on the adaptational strategies to the heat stress in Saccharomyces cerevisiae and the reconstruction of thermotolerance." 京都大学 (Kyoto University), 2017. http://hdl.handle.net/2433/225664.
Full textAbstract:
Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第20439号
農博第2224号
新制||農||1049(附属図書館)
学位論文||H29||N5060(農学部図書室)
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 充美, 教授 間藤 徹, 教授 喜多 恵子
学位規則第4条第1項該当
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Antunes, Catia S. R. "Malaria parasites : oral and nasal inoculation of mice with iRBC's & development of P. falciparum mutants with the CRISPR/cas9 system." Thesis, Bangor University, 2017. https://research.bangor.ac.uk/portal/en/theses/malaria-parasites-oral-and-nasal-inoculation-of-mice-with-irbcs--development-of-p-falciparum-mutants-with-the-crisprcas9-system(bb845996-c41e-4fe7-9d24-17584b9dade8).html.
Full textAbstract:
Malaria is a parasitic disease caused by parasites of the Plasmodium genus and in 2015 around 214,000,000 new cases of malaria infection occurred resulting in 438,000 deaths worldwide. Currently, there is no available vaccine against malaria and despite efforts, mainly channelled into subunit vaccines, immunogenicity is often low. In this work, whole live blood stage Plasmodium was used in oral and nasal inoculation of mice to test for infectivity and immunogenicity. Oral inoculation resulted in infection of some mice while none of those inoculated nasally became infected. Both oral and nasal inoculation, despite not providing protection, allowed for extended survival of inoculated individuals. Given that survival was increased by inoculating animals solely with live parasites, the results detailed in this work show that this approach holds great applicational potential, as coupling oral and nasal inoculation with live parasites and a powerful adjuvant may be the key to unlock full protection. In connection with inoculation of live parasites, this work also aimed at producing a Plasmodium falciparum mutant expressing Salmonella’s FimH+ glycoprotein as to allow its detection by M cells in the gut. This was to be achieved by a modified version of the CRISPR/Cas9 system which would bear a double guide RNA instead of a single. However, this was not possible as the resulting CRISPR plasmid contained an extremely high AT-content becoming toxic to E. coli. In addition to these two main goals, a few molecular techniques crucial for malaria research were improved and a detection method of avian malaria was developed.
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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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Di, Donato Vincenzo. "Axonal target specificity in the CRISPR/Cas9 era : a new role for Reelin in vertebrate visual sytem development." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066409/document.
Full textAbstract:
Les connexions neuronales du système visuel forment des synapses spatialement distribuées en couches discrètes. Comprendre la base du ciblage spécifique axonale est critique pour déchiffrer la formation des réseaux neuronaux complexes. Dans une première étude, nous avons investigué le rôle de la protéine de la matrice extracellulaire Reelin dans la formation in vivo du circuit rétinotectal chez le poisson zèbre. Ce circuit se compose de cellules ganglionnaires de la rétine (CGRs) transmettant l’information visuelle au cerveau via la projection de leur axone dans les différentes couches du tectum optique. Nous avons démontré que la Reelin secrétée par de neurones inhibiteurs localisés dans les couches supérieures du tectum optique forme un gradient. L’induction de mutations délétères dans la voie de signalisation canonicale de la Reelin à l’aide d’outils génétiques a conduit à des défauts de ciblage des axones de CGRs. Nos résultats démontrent un nouveau rôle de la Reelin lors du développement du système visuel et la décrivent comme signature moléculaire nécessaire au ciblage et au positionnement précis des axones de CGRs.Dans une seconde étude, nous avons utilisé la technique CRISPR/Cas9 pour développer une nouvelle approche de mutagénèse conditionnelle chez le poisson zèbre. Nos résultats démontrent que la perturbation de gènes dans des tissues spécifiques peut être effectué par l’induction de l’expression de la protéine Cas9 via le système Gal4/UAS. Nous avons établis un outil pour induire l’apparition de mutations délétères dans des clones de cellules mais aussi dans des cellules individuelles, tous pouvant être suivit distinctement grâce à un marquage génétiqueNeuronal connections in the visual system are arranged in synaptic laminae. Understanding the basis of lamina-specific axonal targeting is critical to gain deeper insights on how complex neural networks form. In a first study we investigated the role of the ECM protein Reelin during zebrafish retinotectal circuit formation in vivo. Here retinal ganglion cells (RGCs) convey the visual information to the brain by projecting their axons to different layers of the optic tectum. We demonstrated that Reelin secreted by a specific class of tectal superficial inhibitory neurons is spatially distributed in a superficial-to-deep gradient within the tectal neuropil. Induced gene disruption for all the components of the canonical Reelin pathway expressed in the retinotectal system resulted in aberrant layering of RGC axons suggesting a role for Reelin pathway in axonal sublaminar segregation. Altogether our findings elucidate a new role for Reelin in vertebrate visual system development, during which it acts as molecular cue by imparting positional information for ingrowing RGCs.In a second study we took advantage of the CRISPR/Cas9 technology to develop a novel approach for conditional mutagenesis in zebrafish. Our results provide evidence that tissue-specific gene disruption can be achieved by driving Cas9 expression with the Gal4/UAS system. We established a tool to induce loss-of-function mutations in cell clones or single cells that can be followed by genetic labeling, enabling their phenotypic analysis. Our technique has the potential to be applied to a wide-range of model organisms, allowing systematic mutagenesis and labeling on a genome-wide scale
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Kennedy, Zachary C. "Optimizing CRISPR/Cas9 for Gene Silencing of SOD1 in Mouse Models of ALS." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1047.
Full textAbstract:
Mutations in the SOD1 gene are the best characterized genetic cause of amyotrophic lateral sclerosis (ALS) and account for ~20% of inherited cases and 1-3% of sporadic cases. The gene-editing tool Cas9 can silence mutant genes that cause disease, but effective delivery of CRISPR-Cas9 to the central nervous system (CNS) remains challenging. Here, I developed strategies using canonical Streptococcus pyogenes Cas9 to silence SOD1. In the first strategy, I demonstrate effectiveness of systemic delivery of guide RNA targeting SOD1 to the CNS in a transgenic mouse model expressing human mutant SOD1 and Cas9. Silencing was observed in both the brain and the spinal cord. In the second strategy, I demonstrate the effectiveness of delivering both guide RNA and Cas9 via two AAVs into the ventricles of the brain of SOD1G93A mice. Silencing was observed in the brain and in motor neurons within the spinal cord. For both strategies, treated mice had prolonged survival when compared to controls. Treated mice also had improvements in grip strength and rotarod function. For ICV treated mice, we detected a benefit of SOD1 silencing using net axonal transport assays, a novel method to detect motor neuron function in mice before onset of motor symptoms. These studies demonstrate that Cas9-mediated genome editing can mediate disease gene silencing in motor neurons and warrants further development for use as a therapeutic intervention for SOD1-linked ALS patients.
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Lindvall, Jenny. "Green and red fluorescent protein tagging of endogenous proteins in glioblastoma using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-314151.
Full textAbstract:
Glioblastoma multiforme is the most malignant primary brain tumor that affects adults, recognized by the World Health Organization as an aggressive grade IV astrocytoma. Patients diagnosed with this type of tumor are left with a poor prognosis even with the most advanced treatment available. The cancer is quite heterogeneous and is typically categorized into four different subtypes depending on genetic aberrations and patient characteristics. Furthermore, researchers have discovered a subpopulation of glioblastoma cells, known as cancer stem cells, which are thought to be resistant to current therapies and responsible for tumor reoccurrence and relapse. Previous studies, in addition to this one, have found that the differentiation of glioblastoma cells downregulate nestin protein expression, the selected stem cell marker, and upregulate glial fibrillary acid protein expression, the selected differentiation marker, using immunofluorescence. Thus, one alternative treatment option is to understand the mechanism underlying the differentiation of cancer stem cells. Four cell cultures representative of each glioblastoma subtype will be endogenously tagged using the genome editing system, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9). The representative stem cell marker, nestin, will be tagged with a green fluorescent protein, while the chosen differentiation marker, glial fibrillary acid protein, will be tagged with a red fluorescent protein. Several drugs were screened to analyze whether the drugs had a differentiation effect on the glioblastoma cells. As a result, strong evidence indicated that bone morphogenetic protein four upregulated glial fibrillary acid protein expression levels to the same extent as the differentiation control media using 5% fetal bovine serum. The goal of this study is to establish a method to directly monitor the differentiation process of glioblastoma cells as a novel molecular screening method. In this case, all glioblastoma cells, even the ones resistant to treatment, can be eliminated through an initial “pre-treatment” by forcing differentiation of cancer stem cells, making the cells more susceptible to the chemotherapy drugs. In the long run, glioblastoma patients would have a chance at a more positive prognosis; a longer life that is free of glioblastoma.Master Thesis in Applied Biotechnology
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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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Stracquadanio, Stefano. "Two strategies to deepen knowledge the antibiotic resistance related traits in Italian MDR Staphylococci: comparative transcriptomic of two DAP-R/DAP-S MRSA isogenic couples and CRISPR system characterization of pathogenic S. epidermidis." Doctoral thesis, Università di Catania, 2019. http://hdl.handle.net/10761/4132.
Full textAbstract:
Antibiotic resistance is worldwide an increasing problem for the public health. Scientists are working to better know the features of Multi-Drug Resistant (MDR) bacteria to develop new strategies to overcome the antibiotic resistance. Among the most common bacteria capable to infect humans and become resistant to antibiotics, the members of the genus Staphylococcus are the etiological agents of some infectious diseases that can be lethal if the pharmacological treatment fails. S. aureus can cause a wide spectrum of diseases, ranging from mild skin forms to systemic forms, whilst S. epidermidis has been frequently implicated in endocarditis and infections of surgical implants and it seems to be the source for antibiotic resistance related gene acquisition by S. aureus.
A comparative transcriptomic analysis by RNA-seq and bioinformatic of two Methicillin-Resistant S. aureus isogenic pairs, the characterization of the CRISPR-Cas system and phage infection sensitivity tests of ten Italian pathogenic clinical MDR S. epidermidis strains were performed to deepen knowledge the antibiotic resistance related traits in Italian MDR Staphylococci.
Results of transcriptomic analysis showed the contribution of Staphylococcal mRNAs and small-RNAs in daptomycin resistance, with multiple pathways associated, including the cell-wall biosynthesis and organization, metabolism, nucleic acid metabolism, stress response and transport, confirming the role of transcriptome in developing antibiotic resistance.
CRISPR-Cas system presence seems to not be a common trait in MDR S. epidermidis (33%) although they showed a high resistance to phage infection, results that should be considered in the perspective of developing a phage therapy.
Finally, recent discoveries suggest the possibility to use CRISPR, in the future, to target not only the genes, but also RNAs (messenger and small), linking transcriptome analysis and CRISPR system studies as parts of a possible strategy to cure the antibiotic resistance.
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Kiritsy, Michael C. "Functional Genomics of Mammalian Innate Immunity." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1102.
Full textAbstract:
The breadth of genetic diversity in the mammalian immune response stands out amongst the ubiquity of variation seen in the genome, evidence that microbial infections have been a major driver of evolution. As technology has facilitated an understanding of the etiology of immunological diversity, so too has it enabled the assessment of its varied functions. Functional genomics, with its ability to assess both cause and effect, has revolutionized our understanding of fundamental biological phenomena and recalibrated our hypotheses. We build upon the model of host immunity established by rare genetic variants that are causative of immunodeficiencies, but that incompletely consider the complexities of the genome. To expand our understanding, we performed a series of forward genetic screens to identify regulators of distinct functions of the innate immune system. Our studies discovered genes with novel functions in antigen presentation and immunoregulation, including several involved in central metabolism. Studies in macrophages and dendritic cells identified mitochondrial respiration as a positive regulator of the interferon-gamma response, and cells incapable of respiration failed to activate T cells. Notably, human mutations in several of these genes are responsible for immune dysfunction. In summary, this work uses new methods in genetic engineering to systematically assess the regulation of innate immunity. Our results suggest that variation in these regulatory pathways is likely to alter immunity in states of health and disease. Thus, our work validates a new approach to identify candidate genes relevant to immune dysfunction.