Dissertations / Theses on the topic 'Gene Editing (CRISPR/Cas9)'
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1
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.
2
Sousa, Maria Cristina Ferreira de. "Targeted gene editing in Neospora caninum using CRISPR/Cas9." Master's thesis, Universidade de Évora, 2021. http://hdl.handle.net/10174/29205.
Abstract:
Apicomplexa are amongst the most prevalent and morbidity-causing pathogen agents
worldwide, representing serious challenges to animal and public health. Neospora caninum
and Besnoitia besnoiti are causing agents of neosporosis and besnoitiosis. Until today, there
are no effective treatment options against these parasitosis. Therefore, it is urgent to invest
in the development of methods for diagnosis, prevention, control, and treatment against these
protozoan pathogens.
The present dissertation is divided in two parts. The first part summarizes three assays
on drug development, testing the in vitro efficacy of selected endochin-like quinolones
(ELQs) against B. besnoiti and N. caninum tachyzoites on a 3-day proliferation inhibition
assay, long-term experiment with the duration of 20 days, and ultrastructural changes induced
by ELQs were evaluated in N. caninum. The second part of the report consists of a
monography reviewing the CRISPR/Cas9 gene editing technology applied to a targeted sag1
gene knock-out in N. caninum assay; Resumo:
Os parasitas do filo Apicomplexa estão entre os agentes patogénicos causadores de
morbilidade mais prevalentes no mundo, representando sérios desafios para a saúde pública
e animal. Neospora caninum e Besnoitia besnoiti são agentes etiológicos da neosporose e
besnoitiose. Até hoje, não existem opções de tratamento e prevenção disponíveis para estas
parasitoses, tornando-se urgente investir no desenvolvimento de métodos para o diagnóstico,
prevenção e tratamento destes protozoários.
A presente dissertação está dividido em duas partes. A primeira parte relativa a três
ensaios focados no desenvolvimento de medicamentos, testa a eficácia in vitro de endoquinas
tipo quinolonas contra taquizoítos de B. besnoiti e N. caninum num ensaio inibiçãoproliferação de três dias, numa experiência de tratamento de longo-curso e através de
microscopia de transmissão de eletrões para avaliar alterações ultraestruturais. A segunda
parte consiste numa monografia sobre a tecnologia de edição genómica CRISPR/Cas9
aplicada ao knock-out do gene sag1 em N. caninum.
3
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.
Abstract:
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.
4
Croci, Susanna. "CRISPR-Cas9 gene editing: a new promising treatment for Rett syndrome." Doctoral thesis, Università di Siena, 2020. http://hdl.handle.net/11365/1120546.
Abstract:
Rett syndrome (RTT) is a neurodevelopmental disorder affecting the central nervous system and is one of the most common causes of intellectual disability in girls, resulting in severe cognitive and physical disabilities. Mutations in MECP2 and FOXG1 genes cause the classic form and the congenital variant of Rett syndrome, respectively. Both genes are transcriptional regulators and both under- and over-expression of these gene cause disease in humans. To characterize the biological mechanisms implicated in disease pathogenesis, we established and characterized a human neuronal model based on genetic reprogramming of patient fibroblasts into induced Pluripotent Stem Cells (iPSCs). Functional analyses performed in MECP2 iPSC-derived neurons demonstrated that these cells closely mimic the impairment of molecular pathway characterizing the disease revealing defects in GABAergic system and cytoskeleton dynamics. Furthermore, we explored the possibility to use iPSC-derived neurons to develop and study a new treatment for RTT patients. Effective therapies are not currently available and the need for tight regulation of MeCP2 and FOXG1 expression for proper brain functioning makes gene replacement therapy risky. Therefore, gene editing would be much more effective. Gene editing based on CRISPR/Cas9 technology and Homology Directed Repair appears an appealing option for the development of new therapeutic approaches. We have engineered a two-plasmid system to correct FOXG1 (c.688C>T (p(Arg230Cys)); C.765G>A (p.Trp255Ter)) and MECP2 (c.473C>T-p.Thr158Met) variants.. Mutation-specific sgRNAs and donor DNAs have been selected and cloned together with an mCherry/GFP reporter system. Cas9 flanked by sgRNA recognition sequences for auto-cleaving has been cloned in a second plasmid. The system has been designed to be ready for in vivo delivery via Adeno-Associated Viral (AAV) vectors. NGS analysis of corrected cells from MECP2 and FOXG1 patients demonstrated an high editing efficiency, ranging from 20 to 80 % of HDR and confirmed that this correction strategy is feasible in neurons. Functional analyses in edited cells confirm the correction of molecular defects due to the mutation. Based on the use of AAV viruses and their capacity to cross the Blood Brain Barrier (BBB) following intravenous injection these experiments will allow us to demonstrate the full potential of gene editing as a therapeutic option for RTT and for other neurodevelopmental disorders currently lacking an effective treatment.
5
Cullot, Grégoire. "Génotoxicité des systèmes CRISPR-Cas9." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0344.
Abstract:
La thérapie génique est une stratégie thérapeutique prometteuse pour le traitement des maladies monogéniques. Si les premières approches, dites additives, ont reposées sur l’utilisation de vecteurs viraux, une part grandissante se tourne désormais vers l’édition génique. Celle-ci est permise par la mise au point de nouvelles générations d’endonucléases, et en particulier le système CRISPR-Cas9. Moins d’une décennie après sa caractérisation, le système CRISPR-Cas9 a permis de faire passer l’édition génique à un stade clinique. Toutefois, dans le même laps de temps, plusieurs interrogations ont été soulevées vis-à-vis de la génotoxicité pouvant être induite par la Cas9. Une littérature émergente pointe le risque de génotoxicité au site ciblé. Le travail de thèse présentée ici s’inscrit dans cette thématique. La première partie de l’étude a eu pour objectif de décrire la génotoxicité induite par une unique cassure double-brin faite par la Cas9. La caractérisation des effets a été faite à la fois à l’échelle nucléotidique, par le suivi de la balance HDR / InDels, mais également à l’échelle du chromosome. Le suivi de l’intégrité chromosomique a permis de mettre en lumière un nouveau risque de génotoxicité encore non-caractérisé. Un système de détection sensible et spécifique de ce risque a été mis au point pour continuer de le caractériser. Le second objectif a été de répondre aux limites soulevées par la génotoxicité non-voulus, en mettant au point une méthode d’édition génique plus sûre et aussi efficace, via l’utilisation d’une unique cassure simple-brin par la Cas9D10A -nickaseGene therapy is a promising therapeutic strategy for the monogenic diseases treatment. If the first approaches, called additive, have relied on the use of viral vectors, a growing share is now turning to gene editing. Less than a decade after its characterization, the CRISPR-Cas9 system has moved gene editing to a clinical stage. However, in the same period of time, several questions have been raised regarding the genotoxicity that can be induced by Cas9. An emerging literature points to the risk of genotoxicity at the targeted site. The thesis work presented here is part of this theme. The first part of the study aimed to describe the genotoxicity induced by a single double-stranded break made by Cas9. Characterization of the effects was done both at the nucleotide level, by monitoring the HDR / InDels balance, but also at the chromosome scale. The monitoring of chromosomal integrity has brought to light a new risk of genotoxicity that was not characterized. A sensitive and specific detection system for this risk has been developed to further characterize it. The second objective was to address the limitations of unwanted genotoxicity by developing a safer and more efficient gene editing method through the use of a single single-stranded breakage by Cas9D10A-nickase
6
Giada, Beligni. "Application of the CRISPR-Cas9 genome editing approach for the correction of the p.Gly2019Ser (c.6055G>A) LRRK2 variant in Parkinson Disease." Doctoral thesis, Università di Siena, 2022. https://hdl.handle.net/11365/1220257.
Abstract:
Parkinson’s disease (PD) is one of the most common long-term degenerative disorders that affect the nervous system. Clinical symptoms are bradykinesia, resting tremor and postural imbalance due to the loss of dopaminergic neurons in the substantia nigra pars compacta. Heterozygous mutations in the Leucine Rich Repeat Kinase 2 gene (LRRK2) have been identified both in familial and sporadic cases of PD. The most common variant is the p.Gly2019Ser substitution (c.6055G>A). To date there is no effective treatment available.
The genome editing tool CRISPR/Cas9 has recently transformed the field of biotechnology and biomedical discovery, posing the basis for the development of innovative treatments. Using CRISPR/Cas9 technology and Homology Directed Repair, our project aims to validate gene editing as an alternative therapeutic approach for PD through the genetic correction of the pathogenic p.Gly2019Ser LRRK2 mutation restoring the wild-type sequence both in human and mouse models. Specifically, we tested various strategies, based on the CRISPR/Cas9-based genome editing technique, for the correction of LRRK2 p.Gly2019Ser (c.6055G>A) variant in primary mouse and human fibroblasts with promising results. If the correction experiments in in vitro models will confirm the good efficiency of the approach, these experiments will represent a fundamental step for the subsequent evaluation of the potential of gene therapy for the treatment of PD as well as other brain disorders for which no therapy is currently available.
7
Poggi, Lucie. "Gene editing approaches of microsatellite disorders : shortening expanded repeats." Electronic Thesis or Diss., Sorbonne université, 2020. http://www.theses.fr/2020SORUS412.
Abstract:
Les maladies à triplet sont dues à des expansions de trinucléotides dans l’ADN. Aucun traitement n’existe pour les soigner. Le but de cette thèse est de mettre au point de nouvelles approches de thérapie génique pour supprimer les expansions pathologiques dans le génome humain. Dans une première partie, un système expérimental dans la levure a été construit afin d’évaluer l’efficacité de différentes nucléases associées au système CRISPR sur des microsatellites. La seconde partie est concentrée sur une maladie à triplet en particulier ; la dystrophie myotonique de type 1 (DM1), qui est due à une expansion d’une répétition de triplets CTG dans la région 3’UTR du gène DMPK. Une nucléase, TALENCTG , construite pour induire une cassure double-brin dans les répétitions CTG en 3’UTR du gène DMPK, induit de manière très efficace des contractions de triplets CTG dans la levure. Des événements de contraction ont été observés lorsque cette nucléase est exprimée. Des expériences in vivo dans un modèle de souris contenant un fragment d’ADN génomique humain de patient contenant 1000 CTG ont été menées. Des particules virales AAV recombinantes portant le gène de la TALEN ont été produites. Après injection intramusculaire, les cellules musculaires expriment la nucléase, mais dû à une toxicité ou immunogénicité de la protéine, l’expression est perdue. Enfin, le système mis au point dans la levure a été transposé dans une lignée cellulaire humaine établie, les HEK293FS. Ce système pourra servir à sélectionner des nucléases actives dans les cellules humainesMicrosatellite disorders are a specific class of human diseases that are due to the expansion of repeated sequences above pathological thresholds. These disorders have varying symptoms and pathogenic mechanisms, caused by the expanded repeat. No cure exists for any of these dramatic conditions. This thesis is investigating new gene editing approaches to remove pathological expansions in the human genome. In a first part, a yeast-based screen was constructed to identify potent CRISPR-associated nucleases that can cut these microsatellites. The second part focuses on myotonic dystrophy type 1 (DM1), which is due to and expanded CTG repeat tract located at the 3’UTR of the DMKP gene. A nuclease, TALENCTG was designed to induce a double strand break into the CTG repeats. It was previously shown to be active in yeast cells, inducing contractions of CTG repeats from a DM1 patient integrated into the yeast genome. The TALEN was tested in DM1 patient cells. The nuclease was found to trigger some contraction events in patient cells. In vivo experiments were carried out in a mouse model of myotonic dystrophy type 1 containing a human genomic fragment from a patient and 1000 CTG. Intramuscular injections of recombinant AAV encoding the TALENCTG revealed that the nuclease is toxic and/or immunogenic in muscle cells in the tested experimental conditions. Finally, the reporter assay integrated in yeast to screen nucleases was transposed in HEK293FS cell line. The integrated cassette contains a CTG expansion from a myotonic dystrophy type 1 patient flanked by two halves of GFP genes. This system would enable to find nucleases active in human cells
8
Waghulde, Harshal B. "Mapping and CRISPR/Cas9 Gene Editing for Identifying Novel Genomic Factors Influencing Blood Pressure." University of Toledo Health Science Campus / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=mco1470402637.
9
Jayavaradhan, Rajeswari. "Optimization of Gene Editing Approaches for Human Hematopoietic Stem Cells." University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1543919940219677.
10
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.
Abstract:
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
11
Amaya, Colina Anais Karime. "Towards the Treatment of Human Genetic Liver Disease by AAV-Mediated Genome Editing and Selective Expansion of Repaired Hepatocytes." Thesis, The University of Sydney, 2019. https://hdl.handle.net/2123/21893.
Abstract:
Gene repair involves the correction of the genetic mutation directly at the defective locus with retention of physiological expression. The biggest challenge of this approach, however, is that gene repair by homologous recombination occurs at levels that are unlikely to be sufficient to confer therapeutic benefit in the majority of cell-autonomous liver disease phenotypes, such as OTC deficiency, the most common urea cycle disorder. To overcome this challenge, gene correction can be complemented by selective expansion strategies designed to expand repaired hepatocytes to frequencies required for therapeutic benefit. In vivo expansion can be achieved, for instance, by conferring a selective advantage to gene-corrected cells. In this study, human-specific genetic inhibitors were designed to exploit a selective expansion strategy based on the modulation of the tyrosine catabolism pathway and were successfully validated in humanised (Fah-/-, Rag2-/-, IL2rg-/-) FRG mice. Another way to increase the frequency of gene repair is to use nucleases to create DNA breaks at the target site to promote homology-directed repair (HDR). Recombinant AAV vectors carrying human-specific reagents for CRISPR/Cas9-mediated genome editing were developed in order to correct a single nucleotide mutation in exon 9 of the OTC gene. Initially, the editing reagents were evaluated in OTC-deficient mice with a transposed engineered “minigene” version of the OTC gene. Editing reagents functionally validated in this model were then evaluated in vivo on the native OTC locus in primary human hepatocytes, including patient-derived hepatocytes, xenografted into FRG mice. Availability of novel synthetic AAV capsids, such as NP59, facilitated high targeting efficiency of human hepatocytes which in turn resulted in up to 29% OTC alleles being corrected by HDR. The studies described in this thesis show for the first time precise gene repair of a disease-causing mutation in primary human hepatocytes in vivo.
12
Campbell, Ian. "Optimization of Methods for Generating Customized Gene-Edited Human Pluripotent Stem Cells." University of Cincinnati / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1504802720510926.
13
Feehan, Joanna Marie. "Development of methodology for genome editing in Xenopus laevis using CRISPR/Cas9, targeting the rhodopsin gene." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/57863.
Abstract:
Xenopus laevis is a commonly used research subject for retinal physiology and cell biology studies, but its utility is limited by the lack of a robust technology for generation of knock-out (KO) or knock-down (KD) phenotypes. However, new genome manipulation techniques involving CRISPR/Cas9 offer an opportunity for generating gene KOs in X. laevis. RNA-guided Cas9 endonuclease introduces double-stranded DNA breaks into the genome, which are either repaired by error-prone non-homologous-end joining (NHEJ), facilitating indel generation, or by less error-prone homology-directed repair (HDR), facilitating insertion of specific sequences. Rhodopsin was targeted for editing as the expected phenotypes, missing/malformed rod photoreceptor outer segments and lower rhodopsin content, are easily assayed. RNA and transgene methods for CRISPR/Cas9-mediated rhodopsin KOs and knock-ins (KI) in rod photoreceptors of X. laevis were tested, and an RNA injection protocol was developed and optimized. KOs were generated by in vitro transcription and microinjection of Cas9 mRNA, eGFP mRNA, and sgRNAs into in vitro fertilized eggs. Cas9 transgene cassettes were built and tested but editing attempts were unsuccessful. Indel mutations were identified by direct sequencing of PCR products and further characterized by sequencing individual clones. The extent of rhodopsin KO was quantified in 14 post-fertilization day-old tadpoles by anti-rod opsin dot blot assay of retinal extracts, and retinal phenotypes were assessed by cryosectioning and immunolabeling contralateral eyes for confocal microscopy. HDR-mediated KIs were generated by co-injection of a DNA repair fragment, with sufficient homology to the genomic region surrounding predicted dsDNA break-site. Heterologous expression of KIs was confirmed by immunohistochemistry. Delivery of Cas9 by RNA injection can produce high frequency homozygous and heterozygous KOs in X. laevis, permitting analysis in the first generation. I was able to obtain extensive KD generating very severe retinal degeneration phenotypes, and germline transmission of Cas9-mediated indels was confirmed. However, KO was never complete. Sequencing results indicate that first generation animals are chimeric containing many independently derived indels. HDR-mediated KI techniques proved possible, but low in efficiency. These techniques significantly advance the utility of X. laevis as an experimental subject for cell biology and physiology studies.Medicine, Faculty of
Graduate
14
Hsu, Patrick David. "Development of the CRISPR nuclease Cas9 for high precision mammalian genome engineering." Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13068392.
Abstract:
Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. To facilitate successful and specific Cas9 targeting, we first optimize the guide RNAs (sgRNA) to significantly enhance gene editing efficiency and consistency. We also systematically characterize Cas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target mutagenesis. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that Cas9-mediated cleavage is unaffected by DNA methylation and that the dosage of Cas9 and sgRNA can be titrated to minimize off-target modification. Additionally, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses. We next demonstrate that Cas9 nickase mutants can be used with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs can reduce off-target activity by over 1,500-fold in human cells. In collaboration with researchers at the University of Tokyo, we further identified a PAM-interacting domain of the Cas9 nuclease that dictates Cas9 target recognition specificity. Finally, we present protocols that provide experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks. Taken together, this work enables a variety of genome engineering applications from basic biology to biotechnology and medicine.
15
Murakami, Yu. "Establishment of a practical gene knock-in system and its application in medaka." Kyoto University, 2020. http://hdl.handle.net/2433/253339.
Abstract:
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
16
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.
Abstract:
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.
17
Corrado, A. "Evaluation of gene editing strategies based on CRISPR/Cas9 to study specific alleles of LGALS3 associated with the risk of papillary thyroid carcinoma." Doctoral thesis, Università di Siena, 2018. http://hdl.handle.net/11365/1040553.
Abstract:
Galectin-3 is involved in thyroid carcinogenesis. The single nucleotide polymorphism
(SNP) rs4644 (c.191C>A, p.Pro64His) within its encoding gene, LGALS3, is
completely conserved across phylogenetically distant organisms, and it was predicted
to be damaging by analysis performed with Polyphen and SIFT. Thus, we
hypothesized that this SNP could affect the risk to develop DTC. The hypothesis was
corroborated by a case-control association study (1155 DTC cases and 1222 controls),
showing a protective role for the carriers of the Histidine-64 (OR=0.66; IC 95% 0.46-
0.93) versus the Proline-64 allele.
To better understand the role of this SNP, cell lines underwent gene editing for
altering the specific LGALS3 genotype.
The “generation” of the cell lines was performed with a Clustered Regularly
Interspaced Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)
system. Different vectors and approaches for DNA strand breaks induction, as well as
alternative strategies of delivery, were employed and compared to each others in order
to identify the most effective in terms of specificity, time, and costs.
Isogenic cell lines were obtained following gene editing although with variable
results, depending from the strategy employed.
Further steps will include the identification of possible off targets and the evaluation
of how global gene expression is affected by Pro to His variation. In the future these
cells will be helpful to understand the biologic role of this aminoacid change within
galectin-3.
18
Prescott, Jack. "Interrogating novel functions of the I kappa B kinases via CRISPR-Cas9 gene editing and small molecule inhibition." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/277025.
Abstract:
The NF-kB signalling pathway is a critical mediator of the cellular responses to inflammatory cytokines. The IκB kinase (IKK) complex, which is composed of two catalytic subunits (IKKα and IKKβ) and one regulatory subunit (IKKγ/NEMO) acts as the master regulator of NF-κB transcription factor activity. Seminal genetic studies in knockout (KO) mouse embryonic fibroblasts (MEFs) have defined two pathways of NF-κB activation; a canonical pathway, activated in response to cytokines such as TNFα/IL-1β, that requires NEMO and predominantly IKKβ catalytic activity; and a non-canonical pathway, activated in response to a subset of TNF-family cytokines, which requires IKKα and NIK kinase. We have generated and validated CRISPR-Cas9 IKKα, IKKβ and IKKα/β DKO HCT116 colorectal cancer cell lines to interrogate novel functions of the I kappa B kinases in colorectal cancer, including the relative contributions of these kinases to the activation of NF-κB signalling pathways downstream of TNFα induction. Contrary to the seminal studies in KO MEFs, IKKα appeared to make a more significant contribution to canonical NF-κB induction in these cells than IKKβ. Western blot studies demonstrated that both IKKs contributed to the phosphorylation and degradation of IκB and the phosphorylation of the NF-κB subunit, p65 at Serine 536. However, high-content immunofluorescence studies demonstrated that IKKα KO cells were defective in TNFα-induced nuclear translocation of p65 compared to WT and IKKβ KO cells. Additionally, NF-κB-driven luciferase reporter assays showed that IKKα, but not IKKβ, KO cells exhibited significantly reduced NF-κB-dependent gene expression following TNFα stimulation. We also have evidence to suggest that the phosphorylation site at Serine 468 on p65, previously defined as an IKKβ-dependent site, is in-fact an IKKα-dependent site in these cells. Furthermore, IKKα knockout revealed a potentially important role for IKKα activity in preventing the stabilisation of NIK protein following prolonged TNFα stimulation. RNA sequencing analysis of wild-type, IKKα KO, IKKβ KO and IKKα/β DKO cells stimulated with TNFα was performed to identify genes whose expression were differentially deregulated by IKK KO. These analyses confirmed the importance of IKKα for canonical NF-κB gene expression. Furthermore, IKKβ knockout had unexpected effects on the expression of a broad range of genes involved in chromatin organisation, cytoskeletal organisation, mitotic cell cycle control and the DNA damage response. During the characterisation of IKK KO cells it was discovered that the expression of NEMO was downregulated at the protein, but not mRNA level by approximately 50% in IKKα KO cells and 90% in IKKα/β DKO cells. IKKβ KO cells, meanwhile, exhibited wild-type NEMO expression. Emetine-chase and radioactive pulse chase labelling experiments demonstrated that the half-life of NEMO in IKKα and IKKα/β DKO cells was significantly shortened due to enhanced proteasomal turnover. Bioinformatics analyses predicted significant regions of intrinsic structural disorder within NEMO, particularly at the N- and C-termini, the former of which overlapped with the IKK binding domain. On this basis, the susceptibility of NEMO to in vitro degradation by the 20S proteasome was examined, with NEMO proving be a highly effective substrate of the 20S proteasome. Importantly, IKKα and IKKβ were both shown to protect NEMO from proteasomal degradation, leading us to propose a model whereby interaction with IKK kinase subunits sequesters/masks intrinsically disordered regions in NEMO that would otherwise make NEMO a highly effective substrate for ubiquitin-dependent and/or ubiquitin-independent proteasomal degradation. BMS-345541 is a commercially available allosteric inhibitor of IKKβ that has been used extensively in numerous studies, including a report that proposed novel functions for IKKβ in mitotic cell cycle progression (Blazkova et al., 2007). Similar antiproliferative effects to those reported by Blazkova et al., were observed during the characterisation of a novel ATP-competitive inhibitor of IKKβ, AZD2230. In depth characterisation of the selectivity of AZD2230 and BMS-345541, however, revealed that the antiproliferative effects of AZD2230 and BMS-345541 are, in fact, due to off-target inhibition, potentially at the level of RNA Polymerase II C-terminal domain phosphorylation, and hence general transcription. Collectively, these studies reveal novel functions of the IKK kinases in NF-κB signalling and inform therapeutic strategies for targeting chronic canonical NF-κB activation in colorectal cancer.
19
Fine, Eli Jacob. "A toolkit for analysis of gene editing and off-target effects of engineered nucleases." Diss., Georgia Institute of Technology, 2015. http://hdl.handle.net/1853/54875.
Abstract:
Several tools were developed to help researchers facilitate clinical translation of the use of engineered nucleases towards their disease gene of interest. Two major issues addressed were the inability to accurately predict nuclease off-target sites by user-friendly \textit{in silico} methods and the lack of a high-throughput, sensitive measurement of gene editing activity at endogenous loci. These objectives were accomplished by the completion of the following specific aims. An online search interface to allow exhaustive searching of a genome for potential nuclease off-target sites was implemented. Previously discovered off-target sites were collated and ranking algorithms developed that preferentially score validated off-target sites higher than other predictions. HEK-293T cells transfected with newly developed TALENs and ZFNs targeting the beta-globin gene were analyzed at the off-target sites predicted by the tool. Many samples of genomic DNA from cells treated with different ZFNs and TALENs were analyzed for off-target effects to generate a greatly expanded training set of bona fide off-target sites. Modifications to the off-target prediction algorithm parameters were evaluated for improvement through Precision-Recall analysis and several other metrics. An analysis pipeline was developed to process SMRT reads to simultaneously measure the rates of different DNA repair mechanisms by directly examining the DNA sequences. K562 cells were transfected with different types of nucleases and donor repair templates in order to optimize conditions for repairing the beta-globin gene. This work will have significant impact on future studies as the methods developed herein allow other laboratories to optimize nuclease-based therapies for single gene disorders.
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Haward, Fiona. "Investigation of the physiological roles of SRSF1-mediated translation." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31188.
Abstract:
The serine/arginine-rich (SR-) family proteins constitute a diverse group of pre-mRNA splicing factors that are essential for viability. They can be characterised based on the presence of one or two RRMs and an RS domain. A subset, of which SRSF1 is the prototype, is capable of nucleocytoplasmic shuttling; a process governed by continual cyclic phosphorylation of the RS domain. In contrast, SRSF2, another member of the SR family, is unable to shuttle due to the presence of a nuclear retention sequence (NRS) at the C-terminus of its RS domain. When this NRS is fused to SRSF1, it prevents nucleocytoplasmic shuttling of the SRSF1-NRS fusion protein. In addition to its nuclear roles, SRSF1 is directly associated with the translation machinery and can activate mRNA translation of target transcripts via an mTOR-dependent mechanism. The specific mRNA translational targets that SRSF1 serves to regulate encode numerous factors including RNA processing factors and cell-cycle proteins. The aim of this work is to study the physiological relevance of SRSF1 cytoplasmic functions, as previous data have relied on overexpression systems. CRISPR/Cas9 editing was used to knock-in the NRS naturally present in SRSF2 at the SRSF1 genomic locus, creating an SRSF1-NRS fusion protein. After numerous attempts, it was only possible to obtain a single viable homozygous clone in mouse embryonic stem cells (mESCs), despite being able to successfully tag the genomic SRSF1 locus. This strongly suggests that the ablation of SRSF1 shuttling ability is highly selected against in mESCs. To assess the physiological importance of SRSF1 nucleocytoplasmic shuttling during development, a mouse model for SRSF1-NRS was also developed. SRSF1-NRS homozygous mice are born at correct Mendelian ratios, but are small in size and present with severe hydrocephalus. Finally, proteomics was used to identify interactors of endogenous cytoplasmic SRSF1 and those that bind the NRS of SRSF2 to gain insights into the mechanism of nuclear retention for non-shuttling SR proteins. In summary, this work analyses the physiological relevance of cytoplasmic SRSF1 function and the consequences of the SRSF1-NRS allele in mouse development.
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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.
Abstract:
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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BONOMELLI, SARA. "L'EDITING GENETICO GERMINALE UMANO, TRA PROBLEMI ETICI E QUESTIONI DI GOVERNANCE." Doctoral thesis, Università degli Studi di Milano, 2022. http://hdl.handle.net/2434/922688.
Abstract:
The dissertation aims at identifying and analyzing the scientific, legal, and ethical issues raised by the perspective of intentional modification of human germline by the potential future use of gene editing techniques in the context of human reproduction.
Such a study makes it possible to formulate some critical considerations about human germline gene editing governance. The dissertation claims that the best option to regulate the use of this biotechnological innovation for reproductive purposes consists of a regulation on a state-by-state-basis, which should however be developed within an international governance framework. Several arguments are suggested to underpin this thesis, and some recent initiatives adhering to such governance pattern are examined.
The research is organized in three chapters.
The first chapter, which is introductive to the real research, focuses on the scientific and technical aspects of the thesis topic. More specifically, this chapter aims at laying the foundations for the subsequent discussion, by defining and explaining the notions of i) DNA, gene, chromosome; ii) genetic mutation and genetic disease; and iii) gene therapy and gene editing. Special attention is paid to this latter technology and especially to its potential use on the human germline. Such use is highly controversial, mainly – but not exclusively – since, unlike modifications made by somatic gene editing, those affecting germinal cells – namely, gametes and zygotes – are transmitted to descendants, and thus to next generations.
The second chapter is divided into two sections. The first section reconstructs and analyses the existing regulations in the field of human germline gene editing at international, supranational and national level, stressing their vagueness, fragmentation and lack of specificity. Given the impossibility of extensively examining all relevant domestic laws, guidelines and policies, those of four countries only – the USA, the UK, China and Italy – have been considered in detail. This choice is motivated by the geographical and cultural representativeness of their respective regulations, as well as by the fact that, except for Italy, those countries conducted nearly all the experiments carried out so far in the field of human germline gene editing. The second section of the chapter precisely focuses on these experiments – both for research and reproductive purposes. Jiankui He’s experiment – which resulted in the birth of the world’s first gene-edited babies in 2018 – and Denis Rebrikov’s germline gene editing clinical trial project are thoroughly described and analyzed.
The third and last chapter deals with the ethical issues raised by the perspective of the potential future implementation of germline gene editing interventions in the context of human reproduction. This chapter too is articulated into two sections. The first section provides the theoretical bases for the subsequent ethical analysis, by dividing the possible future uses of germline gene editing techniques into three categories: i) therapeutic interventions; ii) medical enhancement interventions; and iii) non-medical enhancement interventions. Such categorization is paramount, since the various ethical issues related to human germline gene editing do not always involve all three of these categories, and, even when they do, they tend to carry different connotations according to each category. This becomes clear in the second section of the chapter, which critically explores six main ethically problematic areas related to this biotechnological innovation and their numerous articulations.
Finally, the dissertation argues that the scientific, legal and ethical issues identified and examined throughout the research must be taken into account by proper germline gene editing governance mechanisms, which should be the result of parallel and complementary regulatory initiatives promoted both at national and international level.
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Souza, Gustavo Torres de. "Produção de células MDBK expressando a enzima CAS9 e edição do gene da beta-lactoglobulina pelo sistema CRISPR/Cas9." Universidade Federal de Juiz de Fora (UFJF), 2017. https://repositorio.ufjf.br/jspui/handle/ufjf/6049.
Abstract:
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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
O advento sistema CRISPR/Cas9 tornou o processo de edição gênica consideravelmente mais fácil e direto, uma vez que retirou empecilhos técnicos relacionados aos sistemas já disponíveis. Desta forma, foram permitidos diversos avanços no entendimento da função de elementos genômicos, assim como a produção de embriões geneticamente modificados com diversas finalidades. O atual trabalho objetivou a edição gênica no gene da beta-lactoglobulina em células somáticas bovinas objetivando a produção futura de embriões da espécie geneticamente modificados. Considerando-se que a hipersensibilidade a essa proteína responde pela maior parte das alergias ao leite bovino, a produção de animais cujo leite não contenha essa molécula é de grande interesse para a indústria de laticínios. Durante os experimentos, foi possível obter uma linhagem de células bovinas MDBK expressando a enzima Cas9 (MDBK-Cas). Usando células MDBK e as células MBDK-Cas foi possível se obter com sucesso edições gênicas no locus beta-lactoglobulina utilizando-se os componentes do sistema CRISPR/Cas9 na forma de mRNA da proteína Cas9 e sgRNAs. Conclui-se que o sistema CRISPR/Cas9 pode ser usado com os sgRNA desenhados neste estudo para editar o gene da betalactoglobulina em células MDBK. Assim, células MDBK podem ser utilizadas como alvo o locus em estudo. Modelos de estudos para edição do genoma bovino. Em vista da escassa literatura constando de trabalhos em que tenha sido feita a edição gênica em embriões bovinos, os dados gerados por esse trabalho colaborarão para o avanço do estado da arte no que diz respeito a engenharia gênica de bovinos e no conhecimento do funcionamento do sistema CRISPR/Cas9.
The advent of the CRISPR / Cas9 system made the process of gene editing considerably easier and more straightforward, since it removed technical impediments related to the systems already available. In this way, several advances were made in the understanding of the function of genomic elements, as well as the production of genetically modified embryos for various purposes. The present work aimed at the genetic editing of the beta-lactoglobulin gene in bovine somatic cells aiming at the future production of genetically modified embryos of the species. Considering that hypersensitivity to this protein accounts for most of the allergies to bovine milk, the production of animals whose milk does not contain this molecule is of great interest to the dairy industry. During the experiments, it was possible to obtain a lineage of bovine MDBK cells expressing the Cas9 enzyme (MDBK-Cas). Using MDBK cells and MBDKCas cells it was possible to successfully obtain gene editions at the beta-lactoglobulin locus using the components of the CRISPR / Cas9 system as mRNA of the Cas9 protein and sgRNAs. It is concluded that the CRISPR / Cas9 system can be used with the sgRNAs designed in this study to edit the beta-lactoglobulin gene in MDBK cells. Thus, MDBK cells can be targeted as the locus under study. Models of studies for editing the bovine genome. In view of the scarce literature consisting of studies in which bovine embryos have been genetically engineered, the data generated by this work will contribute to the advancement of the state of the art regarding the genetic engineering of cattle and the knowledge of the functioning of the system CRISPR / Cas9.
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Nejat, Naghmeh. "Gene editing of the representative WRKY family members in an elite malting barley cultivar RGT Planet by CRISPR/Cas9." Thesis, Nejat, Naghmeh (2022) Gene editing of the representative WRKY family members in an elite malting barley cultivar RGT Planet by CRISPR/Cas9. PhD thesis, Murdoch University, 2022. https://researchrepository.murdoch.edu.au/id/eprint/66511/.
Abstract:
Barley is one of the world’s most economically important cereal crops and a superb model plant for genetic studies and elucidation of plant responses to environmental changes and stress-associated events due to its exceptional adaptations to broad climatic and environmental conditions. CRISPR/Cas system has a great potential for crop genetic improvement and has revolutionised genetics research and crop breeding. CRISPR-based gene targeting can accelerate the improvement of market-oriented traits and elite varieties through precision crop breeding. WRKY transcription factors (TFs), as a superfamily of TFs, constitute one of the largest transcription factor families in plants. WRKY TFs with diverse biological functions play a pivotal role in activating several developmental and physiological processes and fine-tuning signalling pathways and defence responses. However, little is known about the exact growth and developmental functions of WRKY family members in barley. In this study, 14 representative WRKY transcription factors were selected from different WRKY groups based on phylogenetic analysis. Pooled gRNAs were synthesised for multiplex CRISPR/Cas9 mutagenesis. gRNA constructs carrying one, two or three gRNA cassettes were assembled and delivered into RGT Planet, a high yielding spring malting barley with superior agronomic traits. A collection of 105 mutant lines with remarkable phenotypic variations were obtained. An average mutation frequency in the T0 population created by the pooled CRISPR/Cas9 was 89.74% containing multiple mutations. This study, for the first time, provided strong evidence of the roles of HvWRKY39 in tiller development and HvWRKY32 in spike development, grain filling, and kernel weight. Evaluation of yield components revealed the contribution of HvWRKY32 and HvWRKY39 toward increasing barley yield potential by approximately 20%. Our findings also demonstrated the roles of HvWRKY TFs in root development and root architecture. These results indicated that TFs have tremendous potential to enhance crop improvement and boost crop yield. This study established a promising genomic editing platform with the modern barley variety RGT Planet. The mutant library generated from this study provides a valuable resource for further detailed functional genomics in barley.
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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.
Abstract:
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.
26
Xu, Huaigeng. "Targeted Disruption of HLA genes via CRISPR-Cas9 generates iPSCs with Enhanced Immune Compatibility." Kyoto University, 2019. http://hdl.handle.net/2433/242420.
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Schneider, Sara Jane. "Delivery of CRISPR/Cas9 RNAs into Blood Cells of Zebrafish: Potential for Genome Editing in Somatic Cells." Thesis, University of North Texas, 2017. https://digital.library.unt.edu/ark:/67531/metadc1011754/.
Abstract:
Factor VIII is a clotting factor found on the intrinsic side of the coagulation cascade. A mutation in the factor VIII gene causes the disease Hemophilia A, for which there is no cure. The most common treatment is administration of recombinant factor VIII. However, this can cause an immune response that renders the treatment ineffective in certain hemophilia patients. For this reason a new treatment, or cure, needs to be developed. Gene editing is one solution to correcting the factor VIII mutation. CRISPR/Cas9 mediated gene editing introduces a double stranded break in the genomic DNA. Where this break occurs repair mechanisms cause insertions and deletions, or if a template oligonucleotide can be provided point mutations could be introduced or corrected. However, to accomplish this goal for editing factor VIII mutations, a way to deliver the components of CRISPR/Cas9 into somatic cells is needed. In this study, I confirmed that the CRISPR/Cas9 system was able to create a mutation in the factor VIII gene in zebrafish. I also showed that the components of CRISPR/Cas9 could be piggybacked by vivo morpholino into a variety of blood cells. This study also confirmed that the vivo morpholino did not interfere with the gRNA binding to the DNA, or Cas9 protein inducing the double stranded break.
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BORRELLI, VIRGINIA MARIA GRAZIA. "Caratterizzazione del gene LIPOSSIGENASI 4 e approccio CRISPR-Cas9 per aumentare la resistenza alla fusariosi di mais." Doctoral thesis, Università Cattolica del Sacro Cuore, 2018. http://hdl.handle.net/10280/53792.
Abstract:
Il Fusarium verticillioides (Fv) causa il marciume rosa della spiga e contamina le cariossidi con fumonisine, una famiglia di micotossine che colpisce mangimi e alimenti considerata cancerogena per l'uomo e gli animali. Sono stati condotti diversi studi per identificare i geni del mais associati alla resistenza della pianta ospite all'infezione da Fv e l'accumulo di fumonisina. È noto che le ossilipine regolano la difesa contro i patogeni e che il cross-talk lipidico ospite-patogeno influenza la patogenesi. A questo proposito, i mutanti di mais trasposonici del gene ZmLOX4, la linea suscettibile W22 e la resistente TZI18 sono stati testati per la resistenza a Fv mediante il saggio biologico Rolled Towel Assay (RTA). Inoltre, sono stati studiati i profili di espressione di 16 geni coinvolti nella via LOX e volatili verdi (GLV) e l'attività della lipossigenasi è stata analizzata nelle stesse linee. Inoltre, è stata applicata la tecnologia di modifica del genoma di Clustered Shortspeed Palindromic Repeat / Cas9 associato (CRISPR / Cas9) regolarmente esaminata per indagare le possibili implicazioni del gene ZmLOX6 e del fattore di trascrizione ZmWRKY125 nei meccanismi di resistenza contro Fv. L'espressione di questi geni è stata precedentemente osservata dagli esperimenti di RNA - Seq in genotipi resistenti al mais e Studi di Genome Wide Association (GWAS) che hanno portato a un SNP significativamente associato a ZmWRKY125. Inoltre, il gene ZmLOX4 è stato overespresso nella linea A188 per valutare un possibile miglioramento della resistenza alla malattia verso Fv. Il lavoro molecolare del CRISPR si basa su una doppia clonazione utilizzando due diverse single guide RNA (sgRNA) per un bersaglio genico. I costrutti sotto il promotore ZmpUBI nel vettore binario p1609 sono stati trasformati nella linea A188 utilizzando la trasformazione mediata da Agrobacterium tumefaciens. Le piante di mais modificate nei geni ZmLOX6 e ZmWRKY125 e ZmLOX4 che sovraesprimono saranno caratterizzate per RTA, prove sperimentali in campo e per il loro contenuto di fumonisina. Inoltre, saranno testati l’attività lipossigenasica totale, i suoi metaboliti derivati e le osslipine, oltre all'analisi dell'espressione dei principali geni coinvolti nella via dell'acido jasmonico.Fusarium verticillioides (Fv) causes ear rot in maize and contaminates the kernels with fumonisins, a family of mycotoxins that affects feed and food and considered carcinogenic for humans and animals. Several studies were conducted to identify maize genes associated with host plant resistance to Fv infection and fumonisin accumulation. It is known that plant lipoxygenase (LOX)-derived oxylipins regulate defense against pathogens and that the host-pathogen lipid cross-talk influences the pathogenesis. In this regard, maize mutants carrying Mu insertions in the ZmLOX4 gene, the susceptible W22 and the resistant TZI18 lines were tested for Fv resistance by the screening method rolled towel assay (RTA). Additionally, the expression profiles of 16 genes involved in the LOX and green leaves volatiles (GLV) pathway were studied and the lipoxygenase activity was investigated in the same lines as well. Furthermore, the genome editing technology of Clustered Regularly Interspaced Short Palindromic Repeat/associated Cas9 (CRISPR/Cas9) was applied in order to investigate the possible implication of the lipoxygenase gene ZmLOX6 and the transcription factor ZmWRKY125 in the resistance mechanisms against Fv. The enhanced expression of these genes was previously observed by RNA - Seq experiments in maize resistant genotypes and Genome Wide Association Studies (GWAS) resulted in one SNP significantly associated with ZmWRKY125. Moreover, the gene ZmLOX4 was over-expressed in the line A188 for evaluating a possible improvement of the disease resistance towards Fv. The CRISPR cloning was based on a double cloning using two different guides (sgRNA) for one gene target. The constructs under the maize promoter ZmpUBI in the binary vector p1609 were transformed into the maize A188 line using Agrobacterium tumefaciens mediated transformation. Maize plants edited in the genes ZmLOX6 and ZmWRKY125, and over-expressing ZmLOX4 will be characterized for Fv resistance using rolled towel assay, field assay and for their fumonisin content. Furthermore, the content of jasmonic acid, its derivative metabolites, and oxylipins will be tested, as well as the expression analysis of the main genes involved in the jasmonic acid pathway will be performed.
29
BORRELLI, VIRGINIA MARIA GRAZIA. "Caratterizzazione del gene LIPOSSIGENASI 4 e approccio CRISPR-Cas9 per aumentare la resistenza alla fusariosi di mais." Doctoral thesis, Università Cattolica del Sacro Cuore, 2018. http://hdl.handle.net/10280/53792.
Abstract:
Il Fusarium verticillioides (Fv) causa il marciume rosa della spiga e contamina le cariossidi con fumonisine, una famiglia di micotossine che colpisce mangimi e alimenti considerata cancerogena per l'uomo e gli animali. Sono stati condotti diversi studi per identificare i geni del mais associati alla resistenza della pianta ospite all'infezione da Fv e l'accumulo di fumonisina. È noto che le ossilipine regolano la difesa contro i patogeni e che il cross-talk lipidico ospite-patogeno influenza la patogenesi. A questo proposito, i mutanti di mais trasposonici del gene ZmLOX4, la linea suscettibile W22 e la resistente TZI18 sono stati testati per la resistenza a Fv mediante il saggio biologico Rolled Towel Assay (RTA). Inoltre, sono stati studiati i profili di espressione di 16 geni coinvolti nella via LOX e volatili verdi (GLV) e l'attività della lipossigenasi è stata analizzata nelle stesse linee. Inoltre, è stata applicata la tecnologia di modifica del genoma di Clustered Shortspeed Palindromic Repeat / Cas9 associato (CRISPR / Cas9) regolarmente esaminata per indagare le possibili implicazioni del gene ZmLOX6 e del fattore di trascrizione ZmWRKY125 nei meccanismi di resistenza contro Fv. L'espressione di questi geni è stata precedentemente osservata dagli esperimenti di RNA - Seq in genotipi resistenti al mais e Studi di Genome Wide Association (GWAS) che hanno portato a un SNP significativamente associato a ZmWRKY125. Inoltre, il gene ZmLOX4 è stato overespresso nella linea A188 per valutare un possibile miglioramento della resistenza alla malattia verso Fv. Il lavoro molecolare del CRISPR si basa su una doppia clonazione utilizzando due diverse single guide RNA (sgRNA) per un bersaglio genico. I costrutti sotto il promotore ZmpUBI nel vettore binario p1609 sono stati trasformati nella linea A188 utilizzando la trasformazione mediata da Agrobacterium tumefaciens. Le piante di mais modificate nei geni ZmLOX6 e ZmWRKY125 e ZmLOX4 che sovraesprimono saranno caratterizzate per RTA, prove sperimentali in campo e per il loro contenuto di fumonisina. Inoltre, saranno testati l’attività lipossigenasica totale, i suoi metaboliti derivati e le osslipine, oltre all'analisi dell'espressione dei principali geni coinvolti nella via dell'acido jasmonico.Fusarium verticillioides (Fv) causes ear rot in maize and contaminates the kernels with fumonisins, a family of mycotoxins that affects feed and food and considered carcinogenic for humans and animals. Several studies were conducted to identify maize genes associated with host plant resistance to Fv infection and fumonisin accumulation. It is known that plant lipoxygenase (LOX)-derived oxylipins regulate defense against pathogens and that the host-pathogen lipid cross-talk influences the pathogenesis. In this regard, maize mutants carrying Mu insertions in the ZmLOX4 gene, the susceptible W22 and the resistant TZI18 lines were tested for Fv resistance by the screening method rolled towel assay (RTA). Additionally, the expression profiles of 16 genes involved in the LOX and green leaves volatiles (GLV) pathway were studied and the lipoxygenase activity was investigated in the same lines as well. Furthermore, the genome editing technology of Clustered Regularly Interspaced Short Palindromic Repeat/associated Cas9 (CRISPR/Cas9) was applied in order to investigate the possible implication of the lipoxygenase gene ZmLOX6 and the transcription factor ZmWRKY125 in the resistance mechanisms against Fv. The enhanced expression of these genes was previously observed by RNA - Seq experiments in maize resistant genotypes and Genome Wide Association Studies (GWAS) resulted in one SNP significantly associated with ZmWRKY125. Moreover, the gene ZmLOX4 was over-expressed in the line A188 for evaluating a possible improvement of the disease resistance towards Fv. The CRISPR cloning was based on a double cloning using two different guides (sgRNA) for one gene target. The constructs under the maize promoter ZmpUBI in the binary vector p1609 were transformed into the maize A188 line using Agrobacterium tumefaciens mediated transformation. Maize plants edited in the genes ZmLOX6 and ZmWRKY125, and over-expressing ZmLOX4 will be characterized for Fv resistance using rolled towel assay, field assay and for their fumonisin content. Furthermore, the content of jasmonic acid, its derivative metabolites, and oxylipins will be tested, as well as the expression analysis of the main genes involved in the jasmonic acid pathway will be performed.
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MINGOIA, MAURA. "Terapia genica della β Talassemia mediante editing del DNA." Doctoral thesis, Università degli Studi di Cagliari, 2016. http://hdl.handle.net/11584/266632.
Abstract:
β (HBB) gene, resulting in absence (β0) or deficiency (β+) of β globin chain synthesis. This genetic disorder occurs most frequently in people from Mediterranean countries, such as Italy. In particular, the data indicates that about 12.6% of the Sardinian subjects are carriers of β thalassemia and these are among the highest frequencies of thalassemia genes found in a Caucasian population. In Sardinia, the disease is generally determined by a nonsense mutation at codon 39 (E39X) of exon 2 causing the interruption of β globin synthesis. Patients homozygous with E39X mutation have a severe anemia and require frequent transfusions and iron chelation. The only definitive cure today possible for β chain hemoglobinopathies is the hematopoietic stem cells transplantation, but it is limited by availability of HLA matched donors. However, in the last few years new therapeutic approaches for this genetic disease are taking place. The correction of disease-causing mutation through the technique of Genome-Editing in patient-specific stem cells and subsequent autologous transplantation would be the ideal approach for the treatment of monogenic diseases such as β thalassemia. However, due to difficulties in obtaining sufficient homologous recombination percentages for therapeutic purposes, the aim of my PhD project is to reproduce artificially the HPFH mutations identified in non-coding regions of the β globin cluster, using the system CRISPR/Cas9 associated with NHEJ pathway. In this way, we hope to restore at therapeutic levels the expression of HBG genes and consequently the synthesis of a functional HbF in order to ameliorate the phenotype of β thalassemia.
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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.
Abstract:
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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Kelterborn, Simon. "Gen-Editierung von Photorezeptorgenen in der Grünalge Chlamydomonas reinhardtii mithilfe des CRISPR/Cas9-Systems." Doctoral thesis, Humboldt-Universität zu Berlin, 2020. http://dx.doi.org/10.18452/21903.
Abstract:
Die Modifikation von Genen ist in den molekularen Biowissenschaften ein fundamentales Werkzeug, um die Funktion von Genen zu studieren (Reverse Genetik). Diese Arbeit hat erfolgreich Zinkfinger- und CRISPR/Cas9-Nukleasen für die Verwendung in C. reinhardtii etabliert, um Gene im Kerngenom gezielt auszuschalten und präzise zu verändern.
Basierend auf vorausgegangener Arbeit mit Zinkfingernukleasen (ZFN) konnte die Transformationseffizienz um das 300-fache verbessert werden, was die Inaktivierung von Genen auch in motilen Wildtyp-Zellen ermöglichte. Damit war es möglich, die Gene für das Kanalrhodopsin-1 (ChR1), Kanalrhodopsin-2 (ChR2) und das Chlamyopsin-1/2-Gen (COP1/2) einzeln und gemeinsam auszuschalten. Eine Analyse der Phototaxis in diesen Stämmen ergab, dass die Phototaxis durch Inaktivierung von ChR1 stärker beeinträchtigt ist als durch Inaktivierung von ChR2.
Um das CRISPR/Cas9-System zu verwenden, wurden die Transformationsbedingungen so angepasst und optimiert, dass der Cas9-gRNA-Komplex als in vitro hergestelltes Ribonukleoprotein in die Zellen transformiert wurde. Um die Bedingungen für präzise Genmodifikationen zu messen und zu verbessern, wurde das SNRK2.2-Gen als Reportergen für eine „Blau-Grün Test“ etabliert. Kleine Insertionen von bis zu 30 bp konnten mit kurzen Oligonukleotiden eingefügt werden, während größere Reportergene (mVenus, SNAP-Tag) mithilfe eines Donor-Plasmids generiert wurden. In dieser Arbeit konnten mehr als 20 nicht-selektierbare Gene – darunter 10 der 15 potenziellen Photorezeptorgene – mit einer durchschnittlichen Mutationsrate von 12,1 % inaktiviert werden.
Insgesamt zeigt diese Arbeit in umfassender Weise, wie Gen-Inaktivierungen und Modifikationen mithilfe von ZFNs und des CRISPR/Cas9-Systems in der Grünalge C. reinhardtii durchgeführt werden können. Außerdem bietet die Sammlung der zehn Photorezeptor-Knockouts eine aussichtsreiche Grundlage, um die Vielfalt der Photorezeptoren in C. reinhardtii zu erforschen.Gene editing is a fundamental tool in molecular biosciences in order to study the function of genes (reverse genetics). This study established zinc-finger and CRISPR/Cas9 nucleases for gene editing to target and inactivate the photoreceptor genes in C. reinhardtii. In continuation of previous work with designer zinc-finger nucleases (ZFN), the transformation efficiency could be improved 300-fold, which enabled the inactivation of genes in motile wild type cells. This made it possible to disrupt the Channelrhodopsin-1 (ChR1), Channelrhodopsin-2 (ChR2) and Chlamyopsin-1/2 (COP1/2) genes individually and in parallel. Phototaxis experiments in these strains revealed that the inactivation of ChR1 had a greater effect on phototaxis than the inactivation of ChR2. To apply the CRISPR/Cas9 system, the transformation conditions were adapted and optimized so that the Cas9-gRNA complex was successfully electroporated into the cells as an in vitro synthesized ribonucleoprotein. This approach enabled gene inactivations with CRISPR/Cas9 in C. reinhardtii. In order to measure and improve the conditions for precise gene modifications, the SNRK2.2 gene was established as a reporter gene for a ‘Blue-Green test’. Small insertions of up to 30 bp were inserted using short oligonucleotides, while larger reporter genes (mVenus, SNAP-tag) were integrated using donor plasmids. Throughout this study, more than 20 non-selectable genes were disrupted, including 10 of the photoreceptor genes, with an average mutation rate of 12,1 %. Overall, this work shows in a comprehensive way how gene inactivations and modifications can be performed in green alga C. reinhardtii using ZFNs or CRISPR/Cas9. In addition, the collection of the ten photoreceptor knockouts provides a promising source to investigate the diversity of photoreceptor genes in C. reinhardtii.
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Zhang, Yuan. "Functional Characterization of Beta-Glucuronosyltransferases (GLCATs) and Hydroxyproline-Galactosyltransferases (GALTs) Involved in Arabinogalactan-Protein (AGP) Glycosylation Using CRISPR/Cas9 Gene Editing Technology In Arabidopsis." Ohio University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1588687871450172.
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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.
Abstract:
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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Hamouri, Fatima. "Contrôle optique de l'activité de protéines et de l'expression de gènes, par photo-activation du cyclofène cagé, pour l’étude de l’initiation du cancer." Electronic Thesis or Diss., Sorbonne université, 2020. http://www.theses.fr/2020SORUS235.
Abstract:
Le poisson-zèbre est devenu un modèle d’étude du cancer de plus en plus répandu au cours des dernières décennies. Alors que la plupart de ces modèles sont générés par l'expression d'oncogènes de mammifères sous des promoteurs tissulaires spécifiques, nous décrivons ici une méthode qui permet un contrôle optique précis de l'activation ou d’inactivation de l’expression de gènes, in vivo, chez le poisson-zèbre. Ainsi, cette méthode permet l’induction de phénotypes tumoraux par l’activation de l’expression constitutive d’un oncogène humain typique, KRASG12V, dans des tissus et des cellules individuelles sélectionnées sans promoteurs tissus-spécifiques, et ce, dans les embryons de poisson-zèbre. Nous démontrons également le contrôle optique de l'expression des oncogènes KRASG12V, CMYC et BRAFV600E ainsi que le contrôle de l’expression et de l’activité du système CRISPR-Cas9. En outre, il convient de noter que la manipulation précise de l'expression des gènes est essentielle pour comprendre la plupart des processus biologiques. De ce fait, notre travail présente une nouvelle approche dont l’objectif est d’initier et d’étudier le cancer chez le poisson-zèbre. De plus, la haute résolution spatio-temporelle de cette méthode en fait un outil précieux pour étudier l'initiation du cancer à partir de cellules uniquesThe zebrafish has become an increasingly popular and valuable cancer model over the past decades. While most of these models are generated by expressing mammalian oncogenes under tissue-specific promoters, here we describe a method that allows for the precise optical control of oncogene expression or inactivation in live zebrafish. Thus, this technique allows for the induction of tumor phenotypes by activating the constitutive expression of a typical human oncogene, KRASG12V, in selected tissues and single cells without tissue-specific promoters in live zebrafish. We also demonstrate the optical control of oncogene expression as KRASG12V, CMYC and BRAFV600E as well as the control of the expression and the activity of the CRISPR-Cas9 system. In addition, it should be noted that accurate manipulation of gene expression is essential to understand most biological processes. Therefore, our work presents a novel approach to initiate and study cancer in zebrafish. Finally, it is also worth noting that the high spatio-temporal resolution of this method makes it a valuable tool for studying cancer initiation from single cells
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Hahn, Florian [Verfasser], Andreas P. M. [Gutachter] Weber, and Peter [Gutachter] Westhoff. "Genome editing and establishment of efficient gene targeting approaches in Arabidopsis using the CRISPR/Cas9 system / Florian Hahn ; Gutachter: Andreas P. M. Weber, Peter Westhoff." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2018. http://d-nb.info/1159373612/34.
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Sürün, Duran [Verfasser], Beatrix [Akademischer Betreuer] Süß, M. Cristina [Akademischer Betreuer] Cardoso, and Harald von [Akademischer Betreuer] Melchner. "High Efficiency Gene Correction in Hematopoietic Cells by Donor Template-free CRISPR/Cas9 Genome Editing / Duran Sürün ; Beatrix Süß, M. Cristina Cardoso, Harald von Melchner." Darmstadt : Universitäts- und Landesbibliothek Darmstadt, 2018. http://d-nb.info/1153546396/34.
38
Yang, Luhan. "Development of Human Genome Editing Tools for the Study of Genetic Variations and Gene Therapies." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11117.
Abstract:
The human genome encodes information that instructs human development, physiology, medicine, and evolution. Massive amount of genomic data has generated an ever-growing pool of hypothesis. Genome editing, broadly defined as targeted changes to the genome, posits to deliver the promise of genomic revolution to transform basic science and personalized medicine. This thesis aims to contribute to this scientific endeavor with a particular focus on the development of effective human genome engineering tools.
39
Ibraheim, Raed R. "Genome Engineering Goes Viral: Repurposing of Adeno-associated Viral Vectors for CRISPR-mediated in Vivo Genome Engineering." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1114.
Abstract:
One of the major challenges facing medicine and drug discovery is the large number of genetic diseases caused by inherited mutations leading to a toxic gain-of-function, or loss-of-function of the disease protein. Microbiology offered a new glimpse of hope to address those disorders with the adaptation of the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) defense system as a genome editing tool. Cas9 is a unique CRISPR-associated endonuclease protein that can be easily programmed with an RNA [a single-guide RNA (sgRNA)] that is complementary to nearly any DNA locus. Cas9 creates a double-stranded break (DSB) that can be exploited to knock out toxic genes or replenish therapeutic expression levels of essential proteins. In addition to a matching sgRNA sequence, Cas9 requires the presence of a short signature sequence [a protospacer adjacent motif (PAM)] flanking the target locus. Over the past few years, several Cas9-based therapeutic platforms have emerged to correct DNA mutations in a wide range of mammalian cell lines, ex vivo, and in vivo by adapting recombinant adeno-associated virus (rAAV). However, most of the applications of Cas9 in the field have been limited to Streptococcus pyogenes (SpyCas9), which, in its wild-type form, suffers from inaccurate editing at off-target sites. It is also difficult to deliver via an all-in-one (sgRNA+Cas9) rAAV approach due to its large size. In this thesis, I describe other Cas9 nucleases and their development as new AAV-based genome editing platforms for therapeutic editing in vivo in mouse disease models. In the first part of this thesis, I develop the all-in-one AAV strategy to deliver a Neisseria meningitidis Cas9 ortholog (Nme1Cas9) in mice to reduce the level of circulating cholesterol in blood. I also help characterize an enhanced Cas9 from another meningococcus strain (Nme2Cas9) and show that it is effective in performing editing not only in mammalian cell culture, but also in vivo by all-in-one AAV delivery. Additionally, I describe two AAV platforms that enable advanced editing modalities in vivo: 1) segmental DNA deletion by delivering two sgRNAs (along with Nme2Cas9) in one AAV, and 2) precise HDR-based repair by fitting Nme2Cas9, sgRNA and donor DNA within a single AAV capsid. Using these tools, we successfully treat two genetic disorders in mice, underscoring the importance of this powerful duo of AAV and Cas9 in gene therapy to advance novel treatment. Finally, I present preliminary data on how to use these AAV.Nme2Cas9 vectors to treat Alexander Disease, a rare progressive neurological disorder. These findings provide a platform for future application of gene editing in therapeutics.
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De, Santis Flavia. "Genome editing to understand neural circuits formation : a novel CRISPR/Cas9-based strategy for conditional mutagenesis and functional study of the role of the meteorin gene family in zebrafish neurodevelopment." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066269/document.
Abstract:
Depuis quelques années, le poisson zèbre (Danio rerio) est devenu un modèle de choix pour l'étude du système nerveux et de ses fonctions. Récemment, des technologies nouvelles d'édition du génome permettent la génération d'allèles mutés de manière constitutionnelle et l'étude fonctionnelle de gènes chez ce modèle vertébré. Néanmoins, certains loci nécessite une inactivation spatiotemporelle précise et contrôlée. La première partie de ma thèse décrit la mise au point d'une nouvelle stratégie de disruption génétique de manière tissu-spécifique, basée sur la technologie du CRISPR/Cas9 et du système UAS/Gal4. Cette technique permet l'introduction de mutations somatiques dans des tissus, des clones ou des cellules individuelles préalablement génétiquement marqués, rendant ainsi possible le suivi in vivo de l'effet de la mutation générée grâce au gène rapporteur. La seconde partie de ma thèse se centre sur l'étude fonctionnelle d'une famille des gènes, les meteorines, durant le développement du système nerveux et lors du ciblage axonale chez le poisson zèbre. Les Meteorines sont des protéines conservées chez les vertébrés qui ont été impliquées dans la prolifération, la différentiation des progéniteurs de neurones et notamment dans l'élongation axonale in vitro. Nous avons pu mettre en évidence que les meteorines sont exprimées le long de la ligne médiane du système nerveux chez les larves et au niveau du plancher de la partie postérieure du cerveau et de la moelle épinière. Par l'utilisation du CRISPR/Cas9, nous avons généré des lignées mutantes pour chaque gène meteorine et avons ainsi procédé à l'analyse de l'établissement des projections axonales dans ces lignées mutantesIn recent years, the zebrafish (Danio rerio) has emerged as a powerful model organism to study neuronal circuit development and function. To date, different genome editing technologies allow the generation of constitutive mutant alleles, permitting the study of gene loss-of-function in this vertebrate model. Nevertheless, to assess the role of certain loci it might be required a precise spatiotemporal control of gene inactivation. The rst part of my thesis describes a novel strategy for tissue-specific gene disruption based on the CRISPR/Cas9 and the Gal4/UAS systems. The described technique allows the induction of somatic mutations in genetically labeled tissues, cell clones or single cells, making it possible to follow the effect of gene disruption in vivo via reporter gene expression. The second part of the thesis focuses on the functional analysis of the role of the meteorin gene family during neuronal development and axonal targeting in zebra sh. Meteorin family is conserved among vertebrates and its members have been shown to be involved in neuronal progenitor proliferation and differentiation and axonal elongation, in vitro. We used the zebrafish nervous system as a model to dissect the role of Meteorins during embryonic development, focusing on their potential role as novel guidance molecules. Interestingly, we found that genes belonging to the meteorin family are expressed along the midline of the larval central nervous system and at the floor plate in the hindbrain and spinal cord. We generated CRISPR/Cas9 mutant lines carrying out-of-frame deletions in the coding sequence of each member of the zebrafish meteorin family and we performed a comprehensive analysis of the establishment of axonal projections in the mutants. Our data pointed out that metrns loss-of-function affects the earliest process of axonal development, demonstrating a crucial role in the process of axonal outgrowth for this new family of evolutionary conserved guidance molecules
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Raper, Austin T. "Mechanistic Studies of DNA Replication, Lesion Bypass, and Editing." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1529576739391675.
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MERCURI, ELISABETTA. "PRECLINICAL MODELING HIGHLIGHTS THE THERAPEUTIC POTENTIAL OF THE ADOPTIVE TRANSPLANT OF GENE CORRECTED T CELLS IN X-LINKED HYPER-IGM IMMUNODEFICIENCY." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2020. http://hdl.handle.net/10281/263922.
Abstract:
La terapia genica di cellule staminali ematopoietiche (HSC) ha prodotto benefici clinici in diversi pazienti affetti da una varietà di malattie genetiche. Tuttavia, l’uso di vettori che si integrano nel genoma in modo semi-casuale pone il rischio di mutagenesi inserzionale e di una espressione del transgene ectopica/non regolata. Quest’ultimo problema è particolarmente rilevante quando si trattano geni strettamente regolati attivi sulla proliferazione cellulare, come il gene CD40LG, la cui espressione sulle cellule T attivate porta all’attivazione contatto-dipendente delle cellule B, alla loro proliferazione ed allo scambio di classe delle immunoglobuline. Poichè le sue mutazioni causano l’immunodeficienza legata all’X con iper-IgM (HIGM1), il trasferimento genico in HSC è stato proposto come potenziale trattamento per questa sindrome. Anche se piccole quantità di cellule trasdotte hanno ripristinato la funzione immunitaria umorale e cellulare in un modello murino di HIGM1, l’espressione costitutiva di CD40LG in timociti o in cellule T periferiche ha portato a linfoproliferazioni, molte delle quali sono progredite a linfom. Le strategie di riparazione genica che preservano il controllo fisiologico dell’espressione del gene corretto potrebbero quindi rappresentare un approccio più promettente per il trattamento di HIGM1. In questo studio sfruttiamo il meccanismo Homology Directed Repair (HDR) per la correzione in situ della maggior parte delle mutazioni responsabili della sindrome HIGM1 presenti nel gene CD40L, con l’obiettivo di ristabilirne la funzione e il controllo dell’espressione. In particolare sfruttiamo il sistema CRISPR/Cas9 per promuovere l’integrazione sito-specifica di una copia funzionale di parte del gene CD40L a valle del suo promotore endogeno, correggendo così la maggior parte delle mutazioni responsabili della malattia. Dato che il difetto genetico non è deleterio per le cellule T, questo tipo di malattia ci offre l’opportunità unica di sviluppare una terapia genica basata sulla correzione di cellule T autologhe. Al fine di stabilire quali sono le dosi terapeutiche e le condizioni di trapianto necessarie per ottenere la ricostituzione immunitaria e il ripristino delle funzioni immunologiche con cellule corrette, abbiamo infuso diverse dosi di cellule T WT in topi HIGM1 pre-condizionati o meno con diversi regimi linfodepletanti ed eseguito trapianti competitivi di cellule staminali ematopoietiche WT e Cd40lg - / - nel modello animale. Mentre l’ analisi del sangue periferico ha dimostrato la persistenza a lungo termine di cellule T in tutte le condizioni, sono stati ottenuti livelli di attecchimento più elevati nei topi trapiantati dopo il trattamento chemioterapico con ciclofosfamide (CPA). Tutti i topi trapiantati hanno mostrato un parziale ripristino della risposta IgG specifica dopo immunizzazione con TNP-KLH, ma è stato osservato un ripristino più elevato nei topi pre-condizionati con CPA. Questi topi hanno anche mostrato la presenza di centri germinativi nella milza. Topi HIGM1 ricostituiti con dosi crescenti di HSPC WT hanno mostrato un ripristino dose-dipendente della risposta immunitaria T dipendente. In particolare, abbiamo dimostrato che il 10% di HSPC funzionali è sufficiente a ripristinare parzialmente la capacità di produrre anticorpi specifici contro diversi antigeni oltre che ad attenuare l'infezione in topi HIGM1 inoculati con il patogeno Pneumocystis murina. Il nostro obiettivo futuro è quello di dimostrare il ripristino della risposta immunitaria contro l'infezione da pneumocystis murina in topi HIGM1 trapiantati con cellule T CD4 + WT. In caso di successo, i nostri risultati saranno strumentali per stabilire il potenziale terapeutico di un approccio di correzione genica basato sulle cellule T per il trattamento della sindrome HIGM1 che potrebbe fungere da terapia ponte per una terapia definitiva basata sul trapianto di HSPC corrette.Background The X-linked hyper-IgM syndrome type I (HIGM1) is caused by inactivating mutations in the CD40 ligand gene (CD40LG) that disrupt the T cell helper function on B cells and macrophages. This disease represents an ideal candidate for a gene correction strategy because preclinical studies of Hematopoietic Stem Cell (HSC) gene therapy have already shown i) evidence of potential efficacy even with few amounts of transduced cells; ii) critical safety issues due to unregulated transgene expression. Since in HIGM1 the genetic defect is not lethal to T cells, we aim to apply our gene editing strategy on autologous T cells that could be used to provide immediate therapeutic benefit to the patients by resolving pre-existing infections prior to a definitive HSPC transplant. Methods To establish which are the therapeutic threshold levels and transplant conditions required to achieve immune reconstitution and functional immunologic restoration with corrected cells, we infused different doses of WT T cells into HIGM1 mice pre-conditioned or not with different lymphodepleting regimens and performed competitive transplants of WT and Cd40lg-/- HSPC in the mouse model. Results While longitudinal blood analyses showed a long-term, stable T cell engraftment in all the conditions, highest engraftment rates were obtained in mice transplanted after chemotherapy treatment with cyclophosphamide (CPA). All the transplanted mice showed a partial rescue of the antigen-specific IgG response after immunization with Keyhole Limpet Hemocyanin (TNP-KLH) but a higher rescue was observed in mice pre-conditioned with CPA. These mice also showed the presence of TNP-KLH specific IgG producing B cells and germinal centers within splenic lymphoid follicles. HIGM1 mice reconstituted with increasing proportions of WT HSPC displayed a dose-dependent rescue of the T cell mediated immune response. In particular we found that 10% of WT HSPC is sufficient to partially restore serologic immunity against different antigens as well as to attenuate infection in HIGM1 mice challenged with Pneumocystis murina. Conclusions Our current efforts are aimed to demonstrate functional restoration of the immune response against Pneumocystis murina infection in HIGM1 mice that received adoptive transfer of WT CD4+ T cells. If successful, our findings will be instrumental to establish the therapeutic potential of a T cell gene correction approach for the treatment of the HIGM1 disease that could act as a bridge therapy to the HSPC-based strategy.
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Moore, Janelle. "Establishment of CRISPR/Cas-9 Aided Knockout of the ZIC2 Gene in the African-American Prostate Cancer Cell Line E006AA-PR." DigitalCommons@Robert W. Woodruff Library, Atlanta University Center, 2019. http://digitalcommons.auctr.edu/cauetds/195.
Abstract:
The largest U.S. cancer health disparity exists in prostate cancer, with African American men having the highest incidence and mortality rates. The present study evaluated the effects of ZIC2 and the underlying mechanisms in the E006 parental African-American cell line that produces tumors at accelerated growth rates because of the increase of ZIC2 genes in African-American males. We analyzed the experimental research that the overexpression of ZIC2 contributes to progression of prostate cancer. E006AA cells with overexpressed or suppressed ZIC2 were analyzed to determine phenotypic differences, PCR, cell proliferation and immunoblot assays. The expression levels of ZIC2 were analyzed by CRISPR-Cas9, Western blot and proliferation growth curves. We discovered using these experimental techniques to knockout ZIC2, reduced cell proliferation occurred. This research investigated the role of ZIC2 in prostate cancer progression and the effects of the loss or gain of function of ZIC2 by using CRISPR-Cas 9 genome editing technology.
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Stephenson, Anthony Aaron. "Mechanistic studies of enzymes involved in DNA transactions." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1531497128385619.
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Martineau, Sabrina. "Etude des mécanismes moléculaires de l'épidermolyse bulleuse simple à partir de cellules souches humaines induites à la pluripotence." Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASQ020.
Abstract:
L'Epidermolyse bulleuse simplex (EBS) est une maladie cutanée principalement causée par des mutations dominantes dans les gènes codant les kératines 5 (KRT5) ou 14 (KRT14). Elle se caractérise notamment par la présence de cloques causées par un décollement de l'épiderme et une inflammation cutanée. D'un point de vue génétique, les mutations vont altérer l'assemblage du réseau de filaments intermédiaires de kératines dans les kératinocytes basaux de l'épiderme et entrainer une cytolyse cellulaire d'où la formation de cloques intra épidermiques. Il n'existe actuellement aucune approche thérapeutique efficace. La compréhension de la maladie et le développement de thérapies, ont été entravées par le manque de modèles cellulaires humains et murins relevant.Ainsi, l'objectif général de ma thèse a consisté à exploiter les propriétés des cellules souches induites à la pluripotence (hiPSc) pour modéliser l'EBS. Dans ce but, nous avons généré des kératinocytes à partir d'hiPSc provenant de patients EBS porteurs de mutations dans le gène KRT5 (Ker-EBS), et de patients sains (Ker-WT). La comparaison des Ker-EBS et Ker-WT nous a permis de montrer que les Ker-EBS récapitulent les principaux phénotypes associés à l'EBS à savoir une diminution de la prolifération cellulaire, une augmentation de la migration cellulaire, une altération des voies de signalisation (ERK et JNK), ainsi que des agrégats de filaments intermédiaires de kératines dans le cytoplasme, tel qu'observé dans kératinocytes primaires de l'EBS. Ces résultats démontrent que notre modèle cellulaire dérivés d'hiPSc est relevant pour l'étude de l'EBS.Afin d'identifier de nouveaux mécanismes moléculaires, une analyse trancriptomique comparant les Ker-EBS aux Ker-WT, a mis en évidence 138 gènes dérégulés, révélant un enrichissement dans les processus liés à la matrice extracellulaire, au packaging de l'ADN et à la réponse inflammatoire. La composante inflammatoire dans l'EBS n'ayant été que peu décrite, la suite de mes travaux a consisté à étudier le phénotype cytokinique pro-inflammatoire. Ainsi, nous avons pu démontrer, une augmentation de l'expression de l'IL-1α, IL-1β, IL-6, IL-8 (CXCL8), CXCL5, CXCL10, CXCL11, CCL5 dans les Ker-EBS, au niveau ARN en condition basale ou stimulée à l'IFNγ pour mimer un contexte pro- inflammatoire. Seules les chemokines CXCL10 et CXCL11 sont secrétées à forte concentration dans le surnagent de culture des Ker-EBS stimulés ou non, démontrant l'implication de ces cytokines dans l'EBS. En parallèle, afin de s'affranchir des biais notamment dus au fond génétique, au sexe, à l'âge des patients et à l'épigénétique, nous avons généré une lignée de Ker-EBS isogénique (Ker-EBS corrigée) par la technique CRISPR-Cas9. Nous avons ainsi pu démontrer que la lignée de Ker-EBS corrigée montrait une restauration du niveau d'expression des cytokines pro-inflammatoires citées précédemment, à un niveau proche des Ker-WT, confirmant un lien direct entre les mutations du gène KRT5 et la signature pro-inflammatoire. Pour conclure, notre nouveau modèle cellulaire nous a permis de reproduire les phénotypes pathologiques connus dans la littérature et de mettre en évidence une dérégulation de l'expression des cytokines pro-inflammatoire dans l'EBS, notamment CXCL10 et CXCL11. Enfin, l'ensemble de ces résultats font de ce modèle un outil pertinent pour permettre une meilleure compréhension des mécanismes moléculaires associés à la pathologie, notamment la composante inflammatoire, ce qui ouvre la voie à de nouvelles approches thérapeutiquesEpidermolysis bullosa simplex (EBS) is a skin disorder caused mainly by dominant mutations in genes coding for keratin 5 (KRT5) or 14 (KRT14) genes. It is characterized by the presence of blisters caused by epidermal detachment, and by other complications such as cutaneous inflammation. From a genetic point of view, the mutations will alter the assembly of the keratin intermediate filament network in basal keratinocytes of the epidermis, leading to cell cytolysis and the formation of intra-epidermal blisters. Currently no effective therapeutic approach it is available. Understanding of the disease and the development of therapies have been hampered by the lack and limitations of relevant human cell and mouse models.So, the general aim of my thesis was to exploit the properties of human induced pluripotent stem cells (hiPSc) to modelling EBS. For this purpose, we generate hiPSc-derived keratinocytes from EBS patients carrying KRT5 mutations (Ker-EBS), and from healthy patients (Ker-WT). Comparison of Ker-EBS and Ker-WT enabled to show that Ker-EBS recapitulates the main phenotypes associated with EBS, namely decreased cell proliferation, increased cell migration, altered signalling pathways (ERK and JNK), as well as aggregates of intermediate keratin filaments in the cytoplasm, as observed in primary EBS keratinocytes. These results demonstrate that our hiPSc-derived cell model is relevant for study EBS.In order to identify new molecular mechanisms, a trancriptomic analysis comparing Ker-EBS with Ker-WT revealed 138 deregulated genes, revealing an enrichment in processes linked to the extracellular matrix, DNA packaging and the inflammatory response. As the inflammatory component in EBS has been poorly described, my next step was to study the pro-inflammatory cytokine phenotype. Thus, we were able to demonstrate increased expression of IL-1α, IL-1β, IL-6, IL-8 (CXCL8), CXCL5, CXCL10, CXCL11, CCL5 in Ker-EBS, at RNA level under basal or IFNy-stimulated conditions to mimic a pro-inflammatory context. Only the chemokines CXCL10 and CXCL11 are secreted at high concentrations in the culture supernatants of stimulated and unstimulated Ker-EBS, demonstrating the involvement of these cytokines in EBS.In parallel, in order to avoid biases due to genetic background, gender, patient age and epigenetics, we generated an isogenic Ker-EBS line (corrected Ker-EBS) using the CRISPR-Cas9 technique. We were thus able to demonstrate that the corrected Ker-EBS line showed a restoration of the expression level of the pro-inflammatory cytokines mentioned above, to a level close to that of Ker-WT, confirming a direct link between mutations in the KRT5 gene and the pro-inflammatory signature.In conclusion, our new cellular model enabled us to reproduce the pathological phenotypes known in the literature, and to demonstrate deregulation of pro-inflammatory cytokine expression in EBS, notably CXCL10 and CXCL11. Taken together, these results make this model a relevant tool to allow a better understanding of the molecular mechanisms associated with the pathology, particularly the inflammatory component, paving the way for new therapeutic approaches
46
Jin, Yehwa. "Research towards the effective disruption of reproductive competence in Nile tilapia Oreochromis niloticus." Thesis, University of Stirling, 2018. http://hdl.handle.net/1893/28382.
Abstract:
Reproductive containment in farmed fish is highly desired for sustainable aquaculture to prevent genetic introgression with wild conspecifics and enhance productivity by suppressing sexual maturation. A number of strategies have already been implemented or have been tested in commercially important fish (e.g. triploidy, monosexing, hormonal therapies); however, they either do not result in 100% containment, or they cannot be applied to all species. One promising new approach consists in disrupting primordial germ cells (PGCs), at the origin of germline cells, to induce sterility. The work carried out in this doctoral thesis aimed to investigate the genes involved in the survival of germ cells and subsequently conduct a functional analysis of candidate genes using CRISPR/Cas9 gene editing system to ultimately provide the basis for the development of a novel sterilisation technique. Nile tilapia was chosen as the experimental animal as it is a major aquaculture species worldwide and the control of reproduction plays a critical role in the farming productivity in this species. In addition, the species has clear advantages as its whole genome sequence is accessible, the generation time is relatively short and zygotes can be available all year round. Initially, a panel of 11 candidate genes with reported roles in survival of PGCs was investigated during the ontogenic development which led to the selection of piwi-like (piwil) gene as a target for genome editing. Then, high temperature was tested as a means to induce germ cell loss to better understand the mechanism underlying germ cell survival and apoptosis, and this study confirmed the functional importance of piwil genes in relation to germ cell loss and proliferation. In addition, the study suggested potential subfunctionalisation within the Bcl-2 gene family which requires further investigation. The next step aimed to optimise the CRISPR/Cas9 gene editing method by improving the microinjection system and testing different concentrations of sgRNAs. Over 95% of injected embryos showed on-target mutation in piwil2 via zygote injection of CRISPR/Cas9 reagents and complete KO larvae were shown in half of the mutants, producing putative sterile fish. However, there was no clear association between the phenotypes in PGCs and the mutation rate. Further comparative studies of mutant screening methods including T7E1, RGEN, HRMA, fragment analysis and NGS revealed that the genotypes of F0 are highly mosaic, suggesting that deep sequencing is recommended for accurate and high throughput F0 screening and further improvement for predictable genome editing is required for a reliable gene functional analysis in F0. In summary, the current thesis provided new scientific knowledge and supporting evidence for the use of the CRISPR/Cas9 gene editing platform to study gene function associated with sterility, with the ultimate goal to develop an alternative sterilisation method in fish.
47
Chai, Shin Luen Chai. "Novel Genetic Modifiers in a Monogenic Cardiac Arrhythmia." Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1516618028568975.
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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.
49
Rodas, Méndez Ana Lucía. "MtSUPERMAN controls the number of flowers per inflorescence and floral organs in the inner three whorls of Medicago truncatula." Doctoral thesis, Universitat Politècnica de València, 2021. http://hdl.handle.net/10251/171474.
Abstract:
[ES] Las leguminosas son un grupo de plantas consideradas de gran importancia por su valor nutricional para la alimentación humana y ganadera. Además, las familias de leguminosas se caracterizan por rasgos distintivos de desarrollo como su inflorescencia compuesta y su compleja ontogenia floral. Para comprender mejor estas características distintivas, es importante estudiar los genes reguladores clave involucrados en el desarrollo de la inflorescencia y la flor. El gen SUPERMAN (SUP) es un factor transcripcional de dedos de zinc (Cys2-Hys2) considerado como un represor activo que controla el número de estambres y carpelos en A. thaliana. Además, SUP está involucrado en la terminación del meristemo floral y el desarrollo de los tejidos derivados del carpelo. El objetivo principal de este trabajo fue la caracterización funcional del ortólogo de SUP en la leguminosa modelo Medicago truncatula (MtSUP). Logramos este objetivo en base a un enfoque de genética reversa, análisis de expresión génica y ensayos de complementación y sobreexpresión. Nuestros resultados muestran que MtSUP es el gen ortólogo de SUP en M. truncatula. MtSUP comparte algunos de los roles ya descritos para SUP con algunas variaciones. Curiosamente, MtSUP controla la determinación del meristemo inflorescente secundario (I2) y de los primordios comunes (CP) a pétalos y estambres. Por tanto, MtSUP controla el número de flores y de pétalos-estambres que producen el meristemo I2 y los primordios comunes, respectivamente. MtSUP muestra funciones novedosas para un gen de tipo SUP, desempeñando papeles clave en los meristemos que confieren complejidad de desarrollo a esta familia de angiospermas. Este trabajo permitió identificar a MtSUP, un gen clave que forma parte de la red reguladora genética que subyace al desarrollo de la inflorescencia compuesta y de las flores en la leguminosa modelo M. truncatula.[CA] Les lleguminoses són un gran grup de plantes considerades de gran importància pel seu valor nutricional per a l'alimentació humana i ramadera. A més, les famílies de lleguminoses es caracteritzen per trets distintius de desenrotllament com la seua inflorescència composta i la seua complexa ontogènia floral. Per a comprendre millor estes característiques distintives, és important estudiar els gens reguladors clau involucrats en la inflorescència i el desenrotllament floral. El gen SUPERMAN (SUP) és un factor transcripcional de dits de zinc (Cys2-Hys2) considerat com un repressor actiu que controla el nombre d'estams i carpels en A. thaliana. A més, SUP està involucrat en la terminació del meristemo floral i el desenrotllament dels teixits derivats del carpel. "L'objectiu principal d'este treball va ser la caracterització funcional de l'ortòleg de SUP en la lleguminosa model Medicago truncatula (MtSUP) . Aconseguim l'objectiu amb base en un enfocament genètic invers, anàlisi d'expressió gènica i assajos de complementació i sobreexpressió. Els nostres resultats mostren que MtSUP és el gen ortòleg de SUP en M. truncatula. MtSUP compartix alguns dels rols ja descrits per a SUP amb variacions. Curiosament, MtSUP està involucrat en la determinació del meristemo de la inflorescència secundària (I2) i els primordios comuns (CP). Per tant, MtSUP controla el nombre de flors i pètals-estams que produïxen el meristemo I2 i els primordios comuns, respectivament. MtSUP mostra funcions noves per a un gen tipus SUP, exercint papers clau en els meristemos que conferixen complexitat de desenrotllament a esta família d'angiospermes. "Este treball va permetre identificar a MtSUP, un gen clau que forma part de la xarxa reguladora genètica darrere de la inflorescència composta i el desenrotllament de flors en la lleguminosa model M. truncatula.
[EN] Legumes are a large group of plants considered of great importance for their nutritional value in human and livestock nutrition. Besides, legume families are characterized by distinctive developmental traits as their compound inflorescence and complex floral ontogeny. For a better understanding of these distinctive features is important to study key regulatory genes involved in the inflorescence and floral development. The SUPERMAN (SUP) gene is a zinc-finger (Cys2-Hys2) transcriptional factor considered to be an active repressor that controls the number of stamens and carpels in A. thaliana. Moreover, SUP is involved in the floral meristem termination and the development of the carpel marginal derived tissues. The main objective of this work was the functional characterization of the SUP orthologue in the model legume Medicago truncatula (MtSUP). We achieved this objective based on a reverse genetic approach, gene expression analysis, and complementation and overexpression assays. Our results show that MtSUP is the orthologous gene of SUP in M. truncatula. MtSUP shares some of the roles already described for SUP with variations. Interestingly, MtSUP controls the determinacy of the secondary inflorescence (I2) meristem and the common primordia (CP). Thus, MtSUP controls the number of flowers and petal-stamens produced by the I2 meristem and the common primordia respectively. MtSUP displays novel functions for a SUP-like gene, playing key roles in the meristems that confer developmental complexity to this angiosperm family. This work allowed to identify MtSUP, a key gene that participates in the genetic regulatory network underlying compound inflorescence and flower development in the model legume M. truncatula.
I would like to thanks the Spanish Ministry of Economy and Competitiveness for the grant (MINECO; BIO2016-75485-R) that supported this work. Special thanks to the Generalitat Valenciana for funding my doctorate with the Santiago Grisolía predoctoral scholarships
Rodas Méndez, AL. (2021). MtSUPERMAN controls the number of flowers per inflorescence and floral organs in the inner three whorls of Medicago truncatula [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171474
TESIS
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Valladares, Rodrigo, and Hanna Briheim. "Metoder och tillämpningar av CRISPR-Cas9 i cancerforskning. : Samt hur CRISPR-Cas9 kan implementeras i skolundervisningen." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-166140.
Abstract:
CRISPR-Cas9 är ett effektivt genredigeringsverktyg som har upptäckts på senare år. Verktyget härstammar från ett adaptivt immunförsvar hos prokaryoter. Tekniken används för att modifiera DNA hos växter, djur och människor på ett enkelt och billigt sätt. CRISPR-Cas9 har visat sig ha stor potential vid bekämpning av olika sjukdomar däribland cancer som idag är ett globalt hälsoproblem. Inom cancerforskningen ses CRISPR-Cas9 som ett lovande verktyg vid cancerterapi och läkemedelsutveckling. I denna studie sammanställer vi aktuella metoder och användningsområden med CRISPR-Cas9 inom cancerforskning. Dessutom undersöker vi hur denna form av genteknik kan lyftas upp och tillämpas i biologiundervisningen.CRISPR-Cas9 has recently emerged as an effective genome editing tool. The tool derives from an adaptive immune system in prokaryotes. The technology is used for modification of DNA in plants, animals and humans in a simple and inexpensive way. CRISPR-Cas9 has shown great potential in fighting different diseases like cancer which today is a global health issue. It is seen as a promising tool for cancer research when it comes to cancer therapy and drug development. Here we summarize current methods and applications of CRISPR-Cas9 for cancer research. Furthermore, we explore the possibilities of introducing and applying this kind of genetic engineering in biology teaching.
Framläggning, opponering och respondering skedde skriftligt till följd av covid19.