Дисертації: "CRISPR, Cas9, genome editing, gRNA"

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.

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2

Ran, Fei Ann. "CRISPR-Cas: Development and applications for mammalian genome editing." Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11610.

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The ability to introduce targeted modifications into genomes and engineer model organisms holds enormous promise for biomedical and technological applications, and has driven the development of tools such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). To facilitate genome engineering in mammalian cells, we have engineered the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 programmable nuclease systems from Streptococcus pyogenes SF370 (SpCas9) and S. thermophilus LMD-9 (St1Cas9) for mouse and human cell gene editing through heterologous expression of the minimal protein and RNA components. We have demonstrated that Cas9 nucleases can be guided by several short RNAs (sgRNAs) to introduce double stranded breaks (DSB) in the mammalian genome and induce efficient, multiplexed gene modification through non-homologous end-joining-mediated indels or homology-directed repair. Furthermore, we have engineered SpCas9 into a nicking enzyme (SpCas9n) to facilitate recombination while minimizing mutagenic DNA repair processes, and show that SpCas9n can be guided by pairs of appropriately offset sgRNAs to induce DSBs with high efficiency and specificity. In collaboration with Drs. Osamu Nureki and Hiroshi Nishimasu at the University of Tokyo, we further report the crystal structure of SpCas9 in complex with the sgRNA and target DNA, and elucidate the structure-function relationship of the ribonucleoprotein complex. Finally, through a metagenomic screen of orthologs, we have identified an additional small Cas9 from Staphylococcus aureus subsp. aureus (SaCas9) that cleaves mammalian endogenous DNA with high efficiency. SaCas9 can be packaged into adeno-associated virus for effective gene modification in vivo. Together, these technologies open up exciting possibilities for applications across basic science, biotechnology, and medicine.

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3

Hirosawa, Moe. "Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch." Kyoto University, 2019. http://hdl.handle.net/2433/242421.

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4

Castanon, velasco Oscar. "Targeting the transposable elements of the genome to enable large-scale genome editing and bio-containment technologies." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLX006.

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Les nucléases programmables et site-spécifiques comme CRISPR-Cas9 sont des signes avant-coureurs d’une nouvelle révolution en génie génétique et portent en germe un espoir de modification radicale de la santé humaine. Le « multiplexing » ou la capacité d’introduire plusieurs modifications simultanées dans le génome sera particulièrement utile en recherche tant fondamentale qu’appliquée. Ce nouvel outil sera susceptible de sonder les fonctions physiopathologiques de circuits génétiques complexes et de développer de meilleures thérapies cellulaires ou traitements antiviraux. En repoussant les limites du génie génétique, il sera possible d’envisager la réécriture et la conception de génomes mammifères. Le développement de notre capacité à modifier profondément le génome pourrait permettre la création de cellules résistantes aux cancers, aux virus ou même au vieillissement ; le développement de cellules ou tissus transplantables compatibles entre donneurs et receveurs ; et pourrait même rendre possible la résurrection d’espèces animales éteintes. Dans ce projet de recherche doctoral, nous présentons l’état de l’art du génie génétique « multiplex », les limites actuelles et les perspectives d’améliorations. Nous tirons profit de ces connaissances ainsi que de l’abondance des éléments transposables de notre ADN afin de construire une plateforme d’optimisation et de développement de nouveaux outils de génie génétique qui autorisent l’édition génomique à grande échelle. Nous démontrons que ces technologies permettent la production de modifications à l’échelle du génome allant jusqu’à 3 ordres de grandeur supplémentaires que précédemment, ouvrant la voie au développement de la réécriture des génomes de mammifères. En outre, l’observation de la toxicité engendrée par la multitude de coupures double-brins dans le génome nous a amenés à développer un bio-interrupteur susceptible d’éviter les effets secondaires des thérapies cellulaires actuelles ou futures. Enfin, en conclusion, nous exposons les potentielles inquiétudes et menaces qu’apporte le domaine génie génétiques et apportons des pistes de réflexions pour diminuer les risques identifiés Programmable and site-specific nucleases such as CRISPR-Cas9 have started a genome editing revolution, holding hopes to transform human health. Multiplexing or the ability to simultaneously introduce many distinct modifications in the genome will be required for basic and applied research. It will help to probe the physio-pathological functions of complex genetic circuits and to develop improved cell therapies or anti-viral treatments. By pushing the boundaries of genome engineering, we may reach a point where writing whole mammalian genomes will be possible. Such a feat may lead to the generation of virus-, cancer- or aging- free cell lines, universal donor cell therapies or may even open the way to de-extinction. In this doctoral research project, I outline the current state-of-the-art of multiplexed genome editing, the current limits and where such technologies could be headed in the future. We leveraged this knowledge as well as the abundant transposable elements present in our DNA to build an optimization pipeline and develop a new set of tools that enable large-scale genome editing. We achieved a high level of genome modifications up to three orders of magnitude greater than previously recorded, therefore paving the way to mammalian genome writing. In addition, through the observation of the cytotoxicity generated by multiple double-strand breaks within the genome, we developed a bio-safety switch that could potentially prevent the adverse effects of current and future cell therapies. Finally, I lay out the potential concerns and threats that such an advance in genome editing technology may be bringing and point out possible solutions to mitigate the risks

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5

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.

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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.

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6

Toffessi, Tcheuyap Vanina. "Development of von Willebrand Factor Zebrafish Mutant Using CRISPR/Cas9 Mediated Genome Editing." Thesis, University of North Texas, 2017. https://digital.library.unt.edu/ark:/67531/metadc984227/.

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von Willebrand factor (VWF) protein acts in the intrinsic coagulation pathway by stabilizing FVIII from proteolytic clearance and at the site of injury, by promoting the adhesion and aggregation of platelets to the exposed subendothelial wall. von Willebrand disease (VWD) results from quantitative and qualitative deficiencies in VWF protein. The variability expressivity in phenotype presentations is in partly caused by the action of modifier genes. Zebrafish has been used as hemostasis animal model. However, it has not been used to evaluate VWD. Here, we report the development of a heterozygote VWF mutant zebrafish using the genome editing CRISPR/Cas9 system to screen for modifier genes involved in VWD. We designed CRISPR oligonucleotides and inserted them into pT7-gRNa plasmid. We then prepared VWF gRNA along with the endonuclease Cas9 RNA from Cas9 plasmid. We injected these two RNAs into 1-4 cell-stage zebrafish embryos and induced a mutation in VWF exon 29 of the zebrafish with a mutagenesis rate of 16.6% (3/18 adult fish). Also, we observed a germline transmission with an efficiency rate of 5.5% (1/18 adult fish). We obtained a deletion in exon 29 which should result in truncated VWF protein.

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7

Canver, Matthew. "Elucidation of Mechanisms of Fetal Hemoglobin Regulation by CRISPR/Cas9 Mediated Genome Editing." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493407.

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Despite nearly complete understanding of the genetics of the β-hemoglobinopathies for several decades, definitive treatment options have lagged behind. Fetal hemoglobin (HbF) reinduction represents a “silver bullet” for therapy of the β-globin disorders. Recent development of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease system has allowed for facile manipulation of the genome for the study of genes and genetic elements. Here we developed CRISPR/Cas9-based methodology to reliably engender targeted genomic deletions ranging from 1.3 kilobases to over 1 megabase, which suggested an inverse relationship between deletion size and deletion frequency. Targeted deletion methods and Cas9-mediated in situ saturating mutagenesis were applied to the enhancer of the HbF repressor BCL11A, which revealed discrete vulnerabilities. This finding is consistent with emerging evidence in the field that large enhancers are comprised of constituent parts with some harboring the majority of the activity. The identified “Achilles heel” of the enhancer represents a promising therapeutic target. We further enhanced the resolution of the in situ saturating mutagenesis technique by using multiple Cas9 nucleases and variant-aware library design to identify functional sequences within the HBS1L-MYB intergenic region, a locus associated with elevated HbF levels. These data demonstrate the robustness of CRISPR/Cas9 mediated in situ saturating mutagenesis and targeted deletion to interrogate functional sequence within regulatory DNA. Harnessing the power of genome editing may usher in a second generation form of gene therapy for the β-globin disorders. Medical Sciences

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8

Antoniani, Chiara. "A genome editing approach to induce fetal hemoglobin expression for the treatment of β-hemoglobinopathies". Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB077.

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Les β-hémoglobinopathies (β-thalassémies et drépanocytose) sont des anémies génétiques qui touchent des milliers de nouveaux nés chaque année dans le monde. Ces maladies sont causées par des mutations affectant l'expression de l'hémoglobine chez l'adulte. Le seul traitement disponible est la transfusion sanguine à vie, associée à une chélation du fer. Pour les patients les plus touchés, la greffe de cellule souche hématopoïétique (CSH) demeure le seul traitement curatif. Néanmoins, la transplantation autologue de cellules souches génétiquement corrigées représente une alternative thérapeutique pour les patients dépourvus de donneur compatible. Certaines délétions naturelles comprenant les gènes de la β- et δ- globine dans le locus de l'hémoglobine sont corrélées à une persistance de l'expression de l'hémoglobine fœtale (HPFH) à l'âge adulte. Ainsi il a été démontré que un taux élevé d'hémoglobine fœtale (HbF) améliore l'évolution clinique de ces deux pathologies. Afin d'identifier les régions régulatrices potentielles de la γ-globine, nous avons combiné les données issues d'analyses de mutations rencontrées chez des patients HPFH avec les sites d'hybridation de facteur de transcription. Sur la base de cette analyse, en ayant recours à la technologie CRISPR/CAS9, nous avons développé un protocole permettant de générer: (i) la délétion d'un potentiel suppresseur de l'HbF situé entre les gènes des globines δ et γ, ciblé par le répresseur de l’HbF BCL11A chez les érythroblastes adultes; (ii) la plus courte délétion associée à des taux élevés d’HbF (délétion Corfu) chez les patients β-thalassemiques; (iii) une délétion de 13.6-kb rencontrée fréquemment chez les patients HPFH et incluant les gènes des globines β et δ ainsi que le potentiel suppresseur de l'HbF. Notre travail a montré que la délétion de la région génomique de 13.6-kb entraîne une forte production de HbF et une réduction concomitante de l'expression de la β-globine soit dans des lignées cellulaires érythroïdes humaines soit dans des érythroblastes primaires dérivées des cellules souches et progéniteurs hématopoïétiques (CSPH). Par ailleurs, nous avons montré que la génération de cette délétion sur des CSPHs issus de patients drépanocytaires entraîne une augmentation de la transcription de la γ-globine dans une proportion significative d'érythroblastes, conduisant à une amélioration du phénotype drépanocytaire. Enfin, nous avons exploré le mécanisme menant à la réactivation de l'expression de la γ-globine. Nous avons évalué des changements dans la conformation de la chromatine et des modifications épigénétiques dans le locus de la β-globine lors de la délétion ou de l'inversion de la région de 13.6 kb. Dans l'ensemble, cette étude contribue à la connaissance des mécanismes favorisant l'échange de l'hémoglobine fœtale à l'adulte et fournit des indices pour une approche d'édition du génome dans le traitement de la β-thalassémies et de la drépanocytose Β-hemoglobinopathies (β-thalassemias and sickle cell disease) are genetic anemias affecting thousands of newborns annually worldwide. β-thalassemias and sickle cell disease (SCD) are caused by mutations affecting the adult hemoglobin expression and are currently treated by red blood cell transfusion and iron chelation regiments. For patients affected by severe β-hemoglobinopathies, allogenic hematopoietic stem cell (HSCs) transplantation is the only definitive therapy. However, transplantation of autologous, genetically corrected HSCs represents an alternative therapy for patients lacking a suitable HSC donor. Naturally occurring large deletions encompassing β- and δ-globin genes in the β-globin gene cluster, defined as Hereditary Persistence of Fetal Hemoglobin (HPFH) traits, lead to increased fetal hemoglobin (HbF) expression ameliorating both thalassemic and SCD clinical phenotypes. In this study, we integrated transcription factor binding site analysis and HPFH genetic data to identify potential HbF silencers in the β-globin locus. Based on this analysis, we designed a CRISPR/Cas9 strategy disrupting: (i) a putative δγ-intergenic HbF silencer targeted by the HbF repressor BCL11A in adult erythroblasts; (ii) the shortest deletion associated with elevated HbF levels (“Corfu” deletion) in β-thalassemic patients, encompassing the putative δγ-intergenic HbF silencer; (iii) a 13.6-kb genomic region including the δ- and β-globin genes and the putative intergenic HbF silencer. Targeting the 13.6-kb region, but not the Corfu and the putative δγ-intergenic regions, caused a robust HbF re-activation and a concomitant reduction in β-globin expression in an adult erythroid cell line and in healthy donor hematopoietic stem/progenitor cells (HSPC)-derived erythroblasts. We provided a proof of principle of this potential therapeutic strategy: disruption of the 13.6-kb region in HSPCs from SCD donors favored the β-to-γ globin switching in a significant proportion of HSPC-derived erythroblasts, leading to the amelioration of the SCD cell phenotype. Finally, we dissected the mechanisms leading to HbF de-repression demonstrating changes in the chromatin conformation and epigenetic modifications within the β-globin locus upon deletion or inversion of the 13.6-kb region. Overall, this study contributes to the knowledge of the mechanisms underlying fetal to adult hemoglobin switching, and provides clues for a genome editing approach to the treatment of SCD and β-thalassemia

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9

Lin, ChieYu. "Characterization and Optimization of the CRISPR/Cas System for Applications in Genome Engineering." Thesis, Harvard University, 2014. http://etds.lib.harvard.edu/hms/admin/view/61.

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The ability to precisely manipulate the genome in a targeted manner is fundamental to driving both basic science research and development of medical therapeutics. Until recently, this has been primarily achieved through coupling of a nuclease domain with customizable protein modules that recognize DNA in a sequence-specific manner such as zinc finger or transcription activator-like effector domains. Though these approaches have allowed unprecedented precision in manipulating the genome, in practice they have been limited by the reproducibility, predictability, and specificity of targeted cleavage, all of which are partially attributable to the nature of protein-mediated DNA sequence recognition. It has been recently shown that the microbial CRISPR-Cas system can be adapted for eukaryotic genome editing. Cas9, an RNA-guided DNA endonuclease, is directed by a 20-nt guide sequence via Watson-Crick base-pairing to its genomic target. Cas9 subsequently induces a double-stranded DNA break that results in targeted gene disruption through non-homologous end-joining repair or gene replacement via homologous recombination. Finally, the RNA guide and protein nuclease dual component system allows simultaneous delivery of multiple guide RNAs (sgRNA) to achieve multiplex genome editing with ease and efficiency. The potential effects of off-target genomic modification represent a significant caveat to genome editing approaches in both research and therapeutic applications. Prior work from our lab and others has shown that Cas9 can tolerate some degree of mismatch with the guide RNA to target DNA base pairing. To increase substrate specificity, we devised a technique that uses a Cas9 nickase mutant with appropriately paired guide RNAs to efficiently inducing double-stranded breaks via simultaneous nicks on both strands of target DNA. As single-stranded nicks are repaired with high fidelity, targeted genome modification only occurs when the two opposite-strand nicks are closely spaced. This double nickase approach allows for marked reduction of off-target genome modification while maintaining robust on-target cleavage efficiency, making a significant step towards addressing one of the primary concerns regarding the use of genome editing technologies. The ability to multiplex genome engineering by simply co-delivering multiple sgRNAs is a versatile property unique to the CRISPR-Cas system. While co-transfection of multiple guides is readily feasible in tissue culture, many in vivo and therapeutic applications would benefit from a compact, single vector system that would allow robust and reproducible multiplex editing. To achieve this, we first generated and functionally validated alternate sgRNA architectures to characterize the structure-function relationship of the Cas9 protein with the sgRNA in DNA recognition and cleavage. We then applied this knowledge towards the development and optimization of a tandem synthetic guide RNA (tsgRNA) scaffold that allows for a single promoter to drive expression of a single RNA transcript encoding two sgRNAs, which are subsequently processed into individual active sgRNAs.

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10

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.

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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.

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11

Bolukbasi, Mehmet F. "Development of Chimeric Cas9 Nucleases for Accurate and Flexible Genome Editing." eScholarship@UMMS, 2017. https://escholarship.umassmed.edu/gsbs_diss/941.

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There has been tremendous amount of effort focused on the development and improvement of genome editing applications over the decades. Particularly, the development of programmable nucleases has revolutionized genome editing with regards to their improvements in mutagenesis efficacy and targeting feasibility. Programmable nucleases are competent for a variety of genome editing applications. There is growing interest in employing the programmable nucleases in therapeutic genome editing applications, such as correcting mutations in genetic disorders. Type II CRISPR-Cas9 bacterial adaptive immunity systems have recently been engineered as RNA-guided programmable nucleases. Native CRISPR-Cas9 nucleases have two stages of sequence-specific target DNA recognition prior to cleavage: the intrinsic binding of the Cas9 nuclease to a short DNA element (the PAM) followed by testing target site complementarity with the programmable guide RNA. The ease of reprogramming CRISPR-Cas9 nucleases for new target sequences makes them favorable genome editing platform for many applications including gene therapy. However, wild-type Cas9 nucleases have limitations: (i) The PAM element requirement restricts the targeting range of Cas9; (ii) despite the presence of two stages of target recognition, wild-type Cas9 can cleave DNA at unintended sites, which is not desired for therapeutic purposes; and (iii) there is a lack of control over the mutagenic editing product that is procuded. In this study, we developed and characterized chimeric Cas9 platforms to provide solutions to these limitations. In these platforms, the DNA-binding affinity of Cas9 protein from S. pyogenes is attenuated such that the target site binding is dependent on a fused programmable DNA-targeting-unit that recognizes a neighboring DNA-sequence. This modification extends the range of usable PAM elements and substantially improves the targeting specify of wild type Cas9. Furthermore, one of the featured chimeric Cas9 variants developed in this study has both robust nuclease activity and ability to generate predictable uniform editing products. These superior properties of the chimeric Cas9 platforms make them favorable for various genome editing applications and bring programmable nucleases one step closer to therapeutic applications.

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12

Amai, Takamitsu. "Development of genome editing technology of mitochondrial DNA in Saccharomyces cerevisiae." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263707.

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13

Jo, Norihide. "Platforms of in vivo genome editing with inducible Cas9 for advanced cancer modeling." Kyoto University, 2019. http://hdl.handle.net/2433/242397.

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14

Fang, Yufeng. "Nuclear Localization of Proteins and Genome Editing in the Oomycete Phytophthora sojae." Diss., Virginia Tech, 2016. http://hdl.handle.net/10919/74232.

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Oomycetes are fungi-like eukaryotic microorganisms, which are actually phylogenetic relatives of diatoms and brown algae, within the kingdom Stramenopila. Many oomycete species, mainly in the genera Phytophthora, Pythium and downy mildews, are devastating plant pathogens that cause multibillion-dollar losses to agriculture annually in the world. Some oomycetes are also animal pathogens, causing severe losses in aquaculture and fisheries, and occasionally causing dangerous infections of humans. Phytophthora species, represented by the Irish Potato Famine pathogen P. infestans and the soybean pathogen P. sojae, are arguably the most destructive pathogens of dicotyledonous plants among the oomycete species and thus have been extensively studied. This dissertation focuses on the model oomycete pathogen P. sojae to investigate specific aspects of its molecular biology and establish an efficient genetic manipulation tool. Specifically, in Chapter 1, I briefly introduce the basic concepts of oomycete biology and pathology, and summarize the experimental techniques used for studies of oomycete genetics over the past two decades. Because the approach to studying fungi and oomycetes are similar (indeed they were incorrectly placed in the same taxonomic group until recently), a special section reviews the emerging genome editing technology CRISPR/Cas system in these organisms together. Chapter 2 and Chapter 3 focus on one of the most important intracellular activities, nuclear localization of proteins, and describe the characterization of nuclear localization signals (NLSs) in P. sojae. This focus stemmed from my early work on genome editing in P. sojae, when I discovered that conventional NLS signals from SV40 used to target the TAL effector nuclease (TALEN) to the nucleus worked poorly in P. sojae. In the first part of this work (Chapter 2), I used confocal microscopy to identify features of nuclear localization in oomycetes that differ from animals, plants and fungi, based on characterization of two classes of nuclear localization signals, cNLS and PY-NLS, and on characterization of several conserved nuclear proteins. In the second part (Chapter 3), I determined that the nuclear localization of the P. sojae bZIP1 transcription factor is mediated by multiple weak nuclear targeting motifs acting together. In Chapter 4 and Chapter 5, I describe my implementation of nuclease-based technology for genetic modification and control of P. sojae. In Chapter 4, I describe the first use of the CRISPR system in an oomycete, including its use to validate the function of a host specificity gene. This is of particular importance because molecular techniques such as gene knockouts and gene replacements, widely used in other organisms, were not previously possible in oomycetes. The successful implementation of CRISPR provides a major new research capability to the oomycete community. Following up on the studies described in Chapter 4, in Chapter 5, I describe the generalization and simplification of the CRISPR/Cas9 expression strategy in P. sojae as well as methods for mutant screening. I also describe several optimized methodologies for P. sojae manipulation based on my 5 years of experience with P. sojae. Ph. D.

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15

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.

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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

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16

Lam, Phuong T. "Crispr/cas9-mediated genome editing of human pluripotent stem cells to advance human retina regeneration research." Miami University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=miami1575372014701457.

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17

Kishimoto, Kenta. "Application of genome editing to marine aquaculture as a new breeding technology." Kyoto University, 2019. http://hdl.handle.net/2433/242704.

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Kyoto University (京都大学) 0048 新制・課程博士 博士(農学) 甲第21827号 農博第2340号 新制||農||1067(附属図書館) 学位論文||H31||N5199(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, 准教授豊原治彦, 准教授田川正朋 学位規則第4条第1項該当

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18

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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19

Mosqueira, Diogo. "Disease modeling hypertrophic cardiomyopathy using CRISPR/Cas9 genome editing technology in human pluripotent stem cell-derived cardiomyocytes." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/51359/.

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

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20

Carstairs, Alice. "Development of in vitro skeletal disease models using CRISPR/Cas9 genome editing in immortalised mesenchymal stem cells." Thesis, University of York, 2017. http://etheses.whiterose.ac.uk/18513/.

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The emergence of engineered nucleases for genome editing has allowed for greater understanding of human biology in health and disease, particularly through combination with stem cells and differentiation protocols. Mesenchymal stem cells (MSCs) are a multipotent adult stem cell able to differentiate into osteoblasts, chondrocytes and adipocytes. Early mesoderm differentiation pathways are relatively well understood, yet the understanding of how mesoderm transcription factors drive post-natal differentiation is less well studied. Additionally, the impact of skeletal disease on MSCs is often neglected in the furthering of our understanding of pathophysiology and disease phenotypes. To this end, this PhD project aimed to use an immortalised MSC cell line (hTERT MSCs) to develop a methodology suitable for the generation of genetically modified MSCs (GM hTERT MSCs). Firstly, the effects of serum in in vitro cell culture was considered by reducing serum in hTERT MSC culture. This demonstrated in the absence of a nutrient-rich environment hTERT MSCs shift towards a lipid-based metabolism with a consequential increase in osteogenic capabilities. The initial targets of CRISPR/Cas9 were Runx2 and Sox9, two critical transcription factors in the onset of osteogenesis and chondrogenesis respectively. The methodology developed used a fluorescent sorting strategy to maximise the possibility of generating GM-hTERT MSCs and in this way, successful genome editing was demonstrated. Genome editing of Runx2 did not appear to absolve osteogenic potential in the hTERT MSCs and targeting of Sox9 via the CRISPR/Cas9 technology demonstrated an apparent increase in adipogenesis. To demonstrate the disease modelling capabilities of GM-hTERT MSCs, a human disease relevant mutation was created in the FGFR3 gene mimicking the genotype of CATSHL syndrome resulting in a striking phenotype, where cells showed a decreased differentiation ability but an increased proliferative and migratory capacity. These data were developed further through the use of a 3D spheroid model allowing for the study of differentiated MSCs, including GM hTERT-MSCs, in a more in vivo like setting. Together these results demonstrate the potential for expanding our understanding of MSC biology in physiologically relevant in vitro conditions.

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21

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/.

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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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22

Vicencio, Jeremy 1990. "Optimizing CRISPR-Cas technologies in Caenorhabditis elegans : Nested CRISPR and expanded targeting with Cas variants." Doctoral thesis, TDX (Tesis Doctorals en Xarxa), 2021. http://hdl.handle.net/10803/672604.

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In this thesis, I present an alternative, cloning-free method for the generation of endogenous fluorescent reporters in the nematode Caenorhabditis elegans. I demonstrate that Nested CRISPR is an efficient method that can be customized for the insertion of a suite of fluorescent tags and epitopes at endogenous loci using a combination of single-stranded and double-stranded DNA repair templates. In this thesis, I also demonstrate the use of enzymes other than Cas9 to target non-NGG PAM sites. The results show that AsCas12a can perform efficient genome editing in TTTV PAMs. Furthermore, the structurally engineered Cas9 variants SpG and SpRY can mediate genome editing in NGN and NYN PAMs, respectively, via both error-prone and precise repair mechanisms under optimized conditions. En esta tesis presento un método alternativo, sin la necesidad de clonaciones moleculares, para la generación de reporteros endógenos fluorescentes en el nematodo Caenorhabditis elegans. Demuestro que Nested CRISPR es una técnica eficiente que puede ser adaptada para la inserción de diversos etiquetas y epítopos fluorescentes en loci endógenos empleando una combinación de moldes de reparación de ADN de cadena simple y doble. También demuestro el uso de enzimas distintas a Cas9 para cubrir secuencias PAM distintas de NGG. Los resultados muestran que AsCas12a puede realizar la edición genómica de manera eficiente en PAMs TTTV. Además, las variantes estructuralmente diseñadas de Cas9, SpG y SpRY, pueden llevar a cabo edición genómica en PAMs NGN y NYN respectivamente, mediante mecanismos propensos a errores y de reparación precisa, en condiciones optimizadas.

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23

Tennant, Peter Andrew. "Genome editing using site-specific nucleases : targeting highly expressed genomic regions for robust transgene expression and genetic analysis." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/22857.

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Integration and expression of exogenous genetic material – in particular, transgenes – into the genomes of model organisms, cell lines or patients is widely used for the creation of genetically modified experimental systems and gene therapy. However, loss of transgene expression due to silencing is still a major hurdle which remains to be overcome. Judicious selection of integration loci can help alleviate the risk of silencing; in recent years the ability to efficiently and specifically target transgene integration has been improved by the advent of site-specific nucleases (SSNs). SSNs can be used to generate double strand breaks (DSBs) in a targeted manner, which increases the efficiency of homologous recombination (HR) mediated transgene integration into predetermined loci. In this work I investigate four human genomic loci for their potential to act as transgene integration sites which will support robust long term expression: the adeno-associated virus (AAV) integration site 1 (AAVS1); the human homologue of the mouse Rosa26 locus (hROSA26); the inosine monophosphate dehydrogenase 2 (IMPDH2) gene and the eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) gene. I also investigate the potential of creating a novel drug-selectable transgene integration system at the IMPDH2 locus to allow for rapid and specific selection of correctly inserted transgenes. In addition to their ability to drive targeted transgene integration, SSNs can be harnessed to specifically disrupt gene function through indel formation following erroneous repair of the induced DSB. Using this strategy, I aimed to answer some important biological questions about eukaryotic translation elongation factor 1 alpha (eEF1A); eEF1A is responsible for providing aminoacylated tRNAs to the ribosome during the elongation phase of protein synthesis. Humans and other vertebrates express two isoforms, eEF1A1 and eEF1A2 (encoded by EEF1A1 and EEF1A2 respectively). During development eEF1A1 is replaced by eEF1A2 in some tissues. The reasons for this remain elusive, but one explanation may lie in the moonlighting functions of eEF1A1, which may not be shared by eEF1A2. Additionally, eEF1A2 can act as an oncogene, while there is no evidence that eEF1A1 is overexpressed in tumours. To begin to untangle these issues I targeted EEF1A1 using SSNs with the aim of making a cell line expressing only the eEF1A2 isoform. This work suggests that eEF1A1 may be essential even in the presence of eEF1A2, though further studies will be required to confirm this.

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24

Vitarelli, Marcela de Oliveira. "Humanização específica do sistema de glicosilação de Pichia pastoris pela técnica CRISPR/Cas9 visando a expressão de glicoproteínas humanas." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-11042017-084657/.

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A produção de proteínas terapêuticas recombinantes compreende moléculas complexas e de alto valor agregado, incluindo a enzima glucocerebrosidase (GCase). Sua deficiência resulta na Doença de Gaucher, passível de tratamento por meio da terapia de reposição enzimática. A forma ativa da GCase recombinante usada na terapia apresenta resíduos terminais de manose expostos no seu perfil de glicosilação. Perfil este que espera-se ser reproduzido por meio da construção de uma linhagem de Pichia pastoris com um padrão de glicosilação humanizado, por meio da deleção de dois genes envolvidos no sistema de glicosilação da levedura: alg3 e och1, responsáveis pela posterior hiper-manosilação característica desse organismo. Assim, a expressão da GCase será usada como modelo no desenvolvimento desta linhagem de Pichia pastoris que permita a expressão de glicoproteínas com um perfil humanizado específico de glicosilação. Além da produção da linhagem mutante pela técnica de CRISPR/Cas9, propomos a construção de duas linhagens controle: uma expressando a proteína GCase para análise do seu padrão selvagem de glicosilação em P. pastoris e outra expressando a proteína Cas9 de Streptoccocus pyogenes (SpCas9). A linhagem P. pastoris/GCase foi construída testando-se duas sequências sinal de secreção diferentes: fosfatase alcalina (PHO1) e albumina humana (Alb). Resultados de western blot mostraram a GCase no lisado celular e baixos níveis de proteína secretada no sobrenadante de cultura, sendo mais expresso na linhagem contendo a sequência PHO1. A linhagem P. pastoris/SpCas9 foi construída e a enzima SpCas9 foi detectada via western blot no lisado celular após indução com metanol. Para a produção da linhagem com padrão de glicosilação humanizado propôs-se a deleção dos genes alg3 e och1 e a inserção, pela via de reparo por recombinação homóloga (HDR), de marcas de resistência aos antibióticos higromicina ou canamicina. Para tal, propusemos a construção de dois vetores finais de expressão do sistema CRISPR/Cas9 em P. pastoris, cada um contendo a enzima SpCas9 e os RNAs guia (gRNAs) para deleção do gene alg3 ou och1, e também a construção de dois fragmentos para HDR contendo o gene de resistência ao antibiótico flanqueado por regiões de 1Kb de homologia com a região de deleção do gene alg3 ou och1. A construção dos vetores e fragmentos para HDR foram inicialmente feitas por meio de técnicas de clonagem clássica. No entanto, apesar de inúmeras tentativas, resultados de PCR e sequenciamento mostraram o insucesso das construções. Partiu-se então para a técnica de Gibson Assembly®, através da qual os dois fragmentos para HDR foram construídos. Porém, os vetores de expressão contendo SpCas9 e os gRNAs ainda apresentam dificuldades na sua construção. Esforços ainda estão sendo feitos para a construção dos vetores e consequente tentativa de estabelecimento das linhagens mutantes. O sucesso no estabelecimento de um sistema de expressão de proteínas heterólogas com este padrão de glicosilação humano específico permitirá a obtenção e possível comercialização da GCase em sua forma terapêutica. Além disso, permitirá possíveis edições genômicas futuras para um padrão de maior complexidade de glicosilação humanizado, criando uma plataforma nacional para produção de outras glicoproteínas terapêuticas de interesse biotecnológico. The production of therapeutic recombinant protein comprises complex and high valued molecules, including the glucocerebrosidase enzyme (GCase). Its deficiency results in Gaucher Disease, susceptible of treatment by enzymatic replacement therapy. The active form of recombinant GCase employed in therapy presents exposed terminal mannose residues in its glycosylation pattern. We hope to reproduce such pattern by constructing a Pichia pastoris strain with a specific human glycosylation pattern through the deletion of two genes involved in yeast glycosylation system, alg3 and och1, responsible for the final hyper-mannosylation characteristic of this organism. Therefore, the expression of GCase will be a case model for the development of the recombinant Pichia pastoris strain that could allow the expression of glycoproteins with a specific humanized glycosylation profile. Despite the establishment of the mutant strain using the CRISPR/Cas9 technique, we propose the construction of two control strains: one expressing the GCase protein for analysis of its wild type glycosylation pattern and another one expressing the Cas9 protein from Streptoccocus pyogenes (SpCas9). The P. pastoris/GCase strain was constructed testing two different secretion signal sequences: alkaline fosfatase (PHO1) and human albumin (Alb). Western blot results have shown GCase in cell lysate and in low expression levels in culture supernatant, being more expressed in the strain containing the PHO1 signal sequence. P. pastoris/SpCas9 strain was constructed and SpCas9 enzyme was detected via western blot in cell lysate after the induction with methanol. To produce the strain with the humanized glycosylation pattern, the deletion of alg3 and och1 genes was proposed along with the insertion, by homology directed repair pathway (HDR), of hygromycin and kanamycin antibiotics resistance marks. In order to do so, we have proposed the construction of two final expression vectors of the CRISPR/Cas9 system in P. pastoris, each one containing SpCas9 enzyme and the guide RNAs (gRNAs) for deletion of alg3 or och1, and also the construction of two fragments for HDR containing the antibiotics resistance gene flanked by 1Kb regions of homology with the deleted regions of alg3 or och1. Vectors and HDR fragments constructions were initially performed using classic cloning techniques. However, despite numerous tries, PCR and sequencing results have shown the failure of the constructions. Then, we moved on to the Gibson Assembly® technique, through which the two HDR fragments were built. Still, the expression vectors containing SpCas9 and the gRNAs presented difficulties in its assembly. Efforts continue to be made to successfully construct the remaining vectors and to establish the mutant lineage. Success in the establishment of a heterologous protein expression system with specific human glycosylation pattern will allow the obtainment and possible commercialization of the therapeutic form of GCase. Furthermore, it will also allow possible future genomic editing to a high complexity human glycosylation pattern, creating a national platform for the production of other therapeutic glycoproteins of biotechnological interest.

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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.

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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.

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26

Edraki, Alireza. "Compact Cas9s and Their Natural Inhibitors for Genome Editing." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1052.

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Recent advances with the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) defense system as genome editing tools have opened a new avenue for targeting disease-causing mutations. The programmability of the Cas9 endonuclease by RNA makes it a potentially powerful therapeutic tool to correct such mutations. The CRISPR-Cas9 system consists of a Cas9 endonuclease that is guided by RNA (sgRNA) to create double-stranded breaks in a target DNA segment complementary to the guide. This process is dependent on a 2-8 nucleotide sequence (called PAM) that is adjacent to the target and functions as a Cas9 binding signal. Each Cas9 ortholog recognizes a unique PAM. However, factors such as the size of Cas9 or the frequency of its PAM sequence in the genome have hindered its clinical use. The Cas9 from Streptococcus pyogenes (SpyCas9) is commonly used in research because its PAM (NGG, where “N” symbolizes any nucleotide) is present every ~8 bp in the genome, providing robust targeting potential. However, it is too large to fit into typical viral vectors used for in vivo delivery, namely adeno-associated vectors (AAV). While several Cas9 orthologs have been characterized, none satisfied the need for a compact, accurate Cas9 with a short PAM. In this thesis, we use two approaches to identify new compact Cas9 orthologs with small PAMs, one using anti-CRISPR proteins and one by searching through closely related Cas9s. First, we use the presence of anti-CRISPRs (naturally occurring, phage-encoded peptides that inhibit CRISPR-Cas9 described in chapter 2) in a genome as indicators of Cas9s that may be highly active. These orthologs come with the added advantage of having inhibitors that can be used as off-switches. We characterize four Cas9s that are targeted by anti-CRISPR proteins and show that they recognize diverse PAMs in vitro. One of the four Cas9’s, namely HpaCas9 from Haemophilus parainfluenzae, induces efficient genome editing in mammalian cells. However, its long N4GATTT PAM does not satisfy the short PAM criterion. For our second approach, we asked whether closely related Cas9 orthologs with drastically different PAM-interacting domains (PIDs, the domain responsible for PAM recognition) recognize different PAMs, and if so, can be used for genome editing. To this end, we exploited natural variation in the PID of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis (Nme2Cas9). Nme2Cas9 recognizes a simple dinucleotide PAM (N4CC) that provides a high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA into adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Finally, we show preliminary data on how CRISPR-Cas9 can be used for therapeutic genome editing for Amytrophic Lateral Sclerosis. Our new findings promise to accelerate the development of genome editing tools for biomedical and therapeutic applications.

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Bressan, Raul Bardini. "Genome editing as a tool to explore transcriptional and epigenetic regulation in neural stem cells and brain cancer." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31095.

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Mammalian neural stem cell (NSC) lines provide a useful experimental model for basic and applied research across stem cell and developmental biology, regenerative medicine and neuroscience. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with functional genetic analyses. However, targeted genetic manipulations have not been reported for mammalian NSC lines. Here, we deploy the CRISPR/Cas9 technology and demonstrate a variety of diverse targeted genetic modifications in both mouse and human NSC lines such as: targeted transgene insertion at safe harbour loci; biallelic knockout of neurodevelopmental genes; knock-in of epitope tags and fluorescent reporters; and engineering of glioma driver mutations at endogenous proto-oncogenes. Leveraging these new optimised methods, we explored gene editing to model the earliest stages of paediatric gliomagenesis in primary human NSCs. Our data indicate that oncogenic mutations in histone H3.3 play a role in NSC transformation and may operate through suppression of replication induced senescence. By comparing cellular responses of NSC cultures from different compartments of the developing brain, we also identify differences in susceptibility to distinct H3.3 mutations that mirror the disease etiology. Altogether, our findings indicate that CRISPR/Cas9-assisted genome editing in NSC lines is a versatile tool to explore gene function in CNS development and cancer biology. Our project resulted in the creation of valuable human cellular models of paediatric gliomagenesis, which will allow us to further our understanding of the disease and carry out cell based drug discovery projects.

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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.

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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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Charpentier, Marine. "Développement de nouvelles approches d’édition du génome à l’aide de nucléases artificielles (TALENs et CRISPR/Cas9)." Thesis, Paris, EPHE, 2016. http://www.theses.fr/2016EPHE3106/document.

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L’édition du génome repose sur la création de cassures double brin à un endroit précis du génome à l’aide de nucléases artificielles (ZFN, TALEN, CRISPR/Cas9) et sur les différents systèmes de réparation que la cellule va mettre en place pour réparer ces dommages. Les deux systèmes de réparation principaux sont le NHEJ (Non Homologous End Joining) et la RH (Recombinaison Homologue). Le NHEJ consiste en une ligation directe des extrémités de la coupure pouvant induire de petites insertions ou délétions avant la ligation. Ces mutations, si elles sont introduites dans un exon, vont modifier le cadre de lecture et pouvoir inactiver le gène cible (Knock Out). La RH permet la réparation de la cassure en recopiant les informations présentes sur la chromatide soeur. Si un ADN exogène comportant des homologies avec la séquence à réparer est inséré avec les nucléases artificielles, la cellule peut le prendre comme matrice de réparation, il est ainsi possible d’insérer n’importe quelle mutation ou transgène de manière précise (Knock In). Ici, différentes stratégies ont été développées pour optimiser ces approches d’édition du génome. Le couplage du domaine Nter de la protéine CtIP à la nucléase Cas9 permet d’augmenter le taux d’insertion par homologie d’un transgène au site de coupure. Le couplage de l’exonucléase Trex2 à la nucléase Cas9 nickase permet quant à lui d’augmenter le taux de mutation après coupure. Ces nouvelles approches peuvent être largement utilisées et permettent de faciliter l’édition du génome Genome editing relies on the ability of artificial nucleases (TALEN or CRISPR/Cas9 system) to induce double strand break into a precise and unique sequence in a whole genome and on the different DNA repair system. The two major DNA repair systems are NHEJ (Non Homologous End Joining) and HR (Homologous Recombination). NHEJ consists on DNA end direct ligation. This system can lead to deletion or insertion at the cut site. These mutations, when induced in an exon, can induce reading frame change and gene inactivation (Knock out). HR consists on the use of sister chromatid to copy lost information in order to complete the double strand break. If an exogenous DNA with homologies with the targeted DNA is inserted with artificial nucleases, it can be used as a template and can permit to introduce any transgene at the cut site (Knock In). In this work, different strategies were used to optimize genome editing. By fusing Nter part of CtIP to Cas9, the KI rate of an exogenous DNA is increased and by fusing Trex2 exonuclease to Cas9, the mutation rate induced is also increased. These two approaches can be widely used to improve genome editing strategies

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30

LLADO, SANTAEULARIA MANEL. "THERAPEUTIC GENOME EDITING IN RETINA AND LIVER." Doctoral thesis, Università degli Studi di Milano, 2020. http://hdl.handle.net/2434/696628.

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In vivo gene therapy with adeno-associated viral (AAV) vectors has been successful at treating several inherited diseases, specifically those caused by loss of function mutations which require transfer of a correct copy of a gene. This would not benefit dominant diseases due to gain of function mutations which produce toxic protein products. In addition, since AAV genomes persist as episomes in target cells, AAV mediated transgene expression might be short lived in tissues where cell proliferation occurs when newborn or after damage, like for example the liver. To overcome these challenges, I have developed AAV-based therapeutic approaches which use genome editing to introduce stable modifications at specific genomic loci. First, an allele-specific approach which targets the Rhodopsin P347S dominant mutation was developed and tested both in vitro and in vivo. I achieved allele-specific targeting of human P347S rhodopsin, which reduced mRNA levels and improved retinal electrical function in a mouse model of autosomal dominant retinitis pigmentosa. Second, I developed a mutation- and homology-independent targeted integration (HITI) approach for gene correction in photoreceptors. I demonstrated feasibility of this approach in mouse and pig photoreceptors using a reporter gene and characterized on-target precision of HITI in the murine rhodopsin locus. I then tested the therapeutic potential of this approach in a mouse model of autosomal dominant retinitis pigmentosa and observed mild and transient improvement of retinal function in treated eyes, which suggests that the levels of editing obtained need optimization. Third, I developed a HITI approach for expressing therapeutic genes from the liver by targeting the albumin locus, which is highly transcribed in hepatocytes. I demonstrated feasibility and efficiency of this approach using a reporter gene, and characterized on-target precision of HITI, as well as off-target integration due to Cas9 cleavage. I then tested the therapeutic potential of the integration of a copy of the human arylsulfatase B (ARSB) gene, which is mutated in a rare lysosomal storage disease, mucopolysaccharidosis type VI (MPS VI), in the albumin locus in the liver of newborn MPSVI mice. I demonstrated that this approach achieves stable expression of ARSB at levels that reduce glucosaminoglycan (GAG) urinary secretion, one of the main readouts of MPSVI phenotype. This stable expression of ARSB is contrary to the decrease of transgene expression observed in neonatal MPSVI mice injected with the same dose of a conventional gene therapy vector, thus overcoming the potential loss of transgene expression caused by hepatocyte proliferation. Overall, I have developed different genome editing approaches for conditions that are inherited as either dominant or recessive. I have tested these approaches in two relevant tissues for gene therapy like retina and liver and shown the potential to provide AAV with persistent transgene expression in proliferating tissues like the newborn liver.

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31

Ballmann, Cora [Verfasser]. "Gezielte Sequenzierung von USP8 bei PatientInnen mit Morbus Cushing und Genome Editing in HAC15 Zellen mittels CRISPR/Cas9 / Cora Ballmann." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2021. http://d-nb.info/1231075147/34.

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32

Kapahnke, Marcel [Verfasser]. "Knock-out of Flotillins in Human Cells Using the CRISPR-Cas9 Genome Editing System: Effects on mRNA Splicing / Marcel Kapahnke." Gießen : Universitätsbibliothek, 2020. http://d-nb.info/1223462137/34.

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33

Stringa, Blerta. "The effect of germline variants on the genesis of early somatic events in cancer explored via Cas9 genome editing." Doctoral thesis, Università degli studi di Trento, 2019. http://hdl.handle.net/11572/242372.

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Although the understanding of genetic predisposition to prostate cancer (PCa) has been improved through genome-wide association studies (GWAS), little is known about the biological implication of germline variants residing in coding or non-coding regions in cancer development and progression. Our hypothesis is that inherited variants may predispose to specific early recurrent genomic events observed in PCa adenocarcinomas, possibly in the context of variable androgen receptor (AR) signaling that changes during a man’s lifetime. Recent in silico analysis by our group on potential association between germline variants and PCa specific somatic lesions identified a non-coding polymorphic regulatory element at the 7p14.3 locus associated with DNA repair and hormone regulated transcript levels and with an early recurrent prostate cancer specific somatic mutation in the Speckle-Type POZ protein (SPOP) gene (OR=5.54, P=1.22e-08) in human prostate tissue data. In order to functionally characterize the polymorphic 7p14.3 locus (rs1376350, single nucleotide polymorphism, G>A), we set up to establish isogenic cell lines harboring the minor allele by using the CRISPR/Cas9 system. In parallel, CRISPR/Cas9 system was used to knock out different portion of the region encompassing the 7p14.3 variant and to eliminate transcription factors (TFs) binding sites that were identified from previous in silico analysis (i.e. AR and CCAAT/Enhancer Binding Protein (C/EBP) beta (CEBPβ)). The transcriptomes of edited pools and edited single clones from macrodeletion (731 bp), microdeletion (50 bp) and alterations of TFs binding sites were analyzed and compared to the transcriptomes of isogenic cells heterozygous (A/G) and homozygous (A/A) for the minor allele A of the risk variant rs1376350 (with or without AR overexpression). These data identified a set of genes scattered throughout the genome with the same pattern of deregulation suggesting the implication of the variant on the regulation of genes residing in different chromosomes. Additionally, ChIP-qPCR experiments for histone modification supported the identification of the 7p14.3 locus with enhancer activity. Furthermore, ChIP-qPCR of histone mark associated with transcriptional activation or repression in isogenic cells harboring the minor allele A upon AR overexpression showed that the activity of the locus is higher for the minor allele A compared to G, independently from AR activation. Despite the limitations of our model and the current lack of validation in other cells, we confirmed that some of the differentially expressed genes that emerged from the comparative analysis of edited cells are deregulated in human normal and tumor prostate samples as well. This work is a proof of concept of germline predisposition to molecularly distinct cancer subclasses and has the potential to nominate new mechanisms of cancer development. Future work aims to elucidate the mechanisms implicated in the deregulation of the transcriptome by combining the information obtained until now with potential new players that we expect to identify by Mass Spectrometry experiments. To clarify the link between the 7p14.3 variant and the somatic mutations in SPOP, we plan to express mutant SPOP in isogenic cells harboring the minor allele and to asses DNA damage response upon overexpression or silencing of TFs binding at and around the rs1376350 variant. My work is an example of how the CRISPR/Cas9 system can be used to develop a technical framework with convergent approaches to functionally characterize polymorphic regulatory regions including but not limited to the establishment of isogenic cells upon single nucleotide editing.

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34

Stringa, Blerta. "The effect of germline variants on the genesis of early somatic events in cancer explored via Cas9 genome editing." Doctoral thesis, Università degli studi di Trento, 2019. http://hdl.handle.net/11572/242372.

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Although the understanding of genetic predisposition to prostate cancer (PCa) has been improved through genome-wide association studies (GWAS), little is known about the biological implication of germline variants residing in coding or non-coding regions in cancer development and progression. Our hypothesis is that inherited variants may predispose to specific early recurrent genomic events observed in PCa adenocarcinomas, possibly in the context of variable androgen receptor (AR) signaling that changes during a man’s lifetime. Recent in silico analysis by our group on potential association between germline variants and PCa specific somatic lesions identified a non-coding polymorphic regulatory element at the 7p14.3 locus associated with DNA repair and hormone regulated transcript levels and with an early recurrent prostate cancer specific somatic mutation in the Speckle-Type POZ protein (SPOP) gene (OR=5.54, P=1.22e-08) in human prostate tissue data. In order to functionally characterize the polymorphic 7p14.3 locus (rs1376350, single nucleotide polymorphism, G>A), we set up to establish isogenic cell lines harboring the minor allele by using the CRISPR/Cas9 system. In parallel, CRISPR/Cas9 system was used to knock out different portion of the region encompassing the 7p14.3 variant and to eliminate transcription factors (TFs) binding sites that were identified from previous in silico analysis (i.e. AR and CCAAT/Enhancer Binding Protein (C/EBP) beta (CEBPβ)). The transcriptomes of edited pools and edited single clones from macrodeletion (731 bp), microdeletion (50 bp) and alterations of TFs binding sites were analyzed and compared to the transcriptomes of isogenic cells heterozygous (A/G) and homozygous (A/A) for the minor allele A of the risk variant rs1376350 (with or without AR overexpression). These data identified a set of genes scattered throughout the genome with the same pattern of deregulation suggesting the implication of the variant on the regulation of genes residing in different chromosomes. Additionally, ChIP-qPCR experiments for histone modification supported the identification of the 7p14.3 locus with enhancer activity. Furthermore, ChIP-qPCR of histone mark associated with transcriptional activation or repression in isogenic cells harboring the minor allele A upon AR overexpression showed that the activity of the locus is higher for the minor allele A compared to G, independently from AR activation. Despite the limitations of our model and the current lack of validation in other cells, we confirmed that some of the differentially expressed genes that emerged from the comparative analysis of edited cells are deregulated in human normal and tumor prostate samples as well. This work is a proof of concept of germline predisposition to molecularly distinct cancer subclasses and has the potential to nominate new mechanisms of cancer development. Future work aims to elucidate the mechanisms implicated in the deregulation of the transcriptome by combining the information obtained until now with potential new players that we expect to identify by Mass Spectrometry experiments. To clarify the link between the 7p14.3 variant and the somatic mutations in SPOP, we plan to express mutant SPOP in isogenic cells harboring the minor allele and to asses DNA damage response upon overexpression or silencing of TFs binding at and around the rs1376350 variant. My work is an example of how the CRISPR/Cas9 system can be used to develop a technical framework with convergent approaches to functionally characterize polymorphic regulatory regions including but not limited to the establishment of isogenic cells upon single nucleotide editing.

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35

Murakami, Yu. "Establishment of a practical gene knock-in system and its application in medaka." Kyoto University, 2020. http://hdl.handle.net/2433/253339.

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

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36

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.

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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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37

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.

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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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38

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.

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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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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.

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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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40

Janin, Grajcarek. "Genome-wide microhomologies enable precise template-free editing of biologically relevant deletion mutations." Kyoto University, 2020. http://hdl.handle.net/2433/253215.

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41

Foster, Robert Graham. "Development of a modular in vivo reporter system for CRISPR-mediated genome editing and its therapeutic applications for rare genetic respiratory diseases." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/33040.

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Rare diseases, when considered as a whole, affect up to 7% of the population, which would represent 3.5 million individuals in the United Kingdom alone. However, while 'personalised medicine' is now yielding remarkable results using recent sequencing technologies in terms of diagnosing genetic conditions, we have made much less headway in translating this patient information into therapies and effective treatments. Even with recent calls for greater research into personalised treatments for those affected by a rare disease, progress in this area is still severely lacking, in part due to the astronomical cost and time involved in bringing treatments to the clinic. Gene correction using the recently-described genome editing technology CRISPR/Cas9, which allows precise editing of DNA, offers an exciting new avenue of treatment, if not cure, for rare diseases; up to 80% of which have a genetic component. This system allows the researcher to target any locus in the genome for cleavage with a short guide-RNA, as long as it precedes a highly ubiquitous NGG sequence motif. If a repair sequence is then also provided, such as a wild-type copy of the mutated gene, it can be incorporated by homology-directed repair (HDR), leading to gene correction. As both guide-RNA and repair template are easily generated, whilst the machinery for editing and delivery remain the same, this system could usher in the era of 'personalised medicine' and offer hope to those with rare genetic diseases. However, currently it is difficult to test the efficacy of CRISPR/Cas9 for gene correction, especially in vivo. Therefore, in my PhD I have developed a novel fluorescent reporter system which provides a rapid, visual read-out of both non-homologous end joining (NHEJ) and homology-directed repair (HDR) driven by CRISPR/Cas9. This system is built upon a cassette which is stably and heterozygously integrated into a ubiquitously expressed locus in the mouse genome. This cassette contains a strong hybrid promoter driving expression of membrane-tagged tdTomato, followed by a strong stop sequence, and then membrane-tagged EGFP. Unedited, this system drives strong expression of membrane-tdTomato in all cell types in the embryo and adult mouse. However, following the addition of CRISPR/Cas9 components, and upon cleavage, the tdTomato is rapidly excised, resulting via NHEJ either in cells without fluorescence (due to imperfect deletions) or with membrane-EGFP. If a repair template containing nuclear tagged-EGFP is also supplied, the editing machinery may then use the precise HDR pathway, which results in a rapid transition from membrane-tdTomato to nuclear- EGFP. Thereby this system allows the kinetics of editing to be visualised in real time and allows simple scoring of the proportion of cells which have been edited by NHEJ or corrected by HDR. It therefore provides a simple, fast and scalable manner to optimise reagents and protocols for gene correction by CRISPR/Cas9, especially compared to sequencing approaches, and will prove broadly useful to many researchers in the field. Further to this, I have shown that methods which lead to gene correction in our reporter system are also able to partially repair mutations found in the disease-causing gene, Zmynd10; which is implicated in the respiratory disorder primary ciliary dyskinesia (PCD), for which there is no effective treatment. PCD is an autosomal-recessive rare disorder affecting motile cilia (MIM:244400), which results in impaired mucociliary clearance leading to neonatal respiratory distress and recurrent airway infections, often progressing to lung failure. Clinically, PCD is a chronic airway disease, similar to CF, with progressive deterioration of lung function and lower airway bacterial colonization. However, unlike CF which is monogenic, over 40 genes are known to cause PCD. The high genetic heterogeneity of this rare disease makes it well suited to such a genome editing strategy, which can be tailored for the correction of any mutated locus.

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42

Marco, Giménez Andrés 1993. "Generation and validation of a CRISPR platform for rapid and inducible genome editing in human pluripotent stem cells and kidney organoids." Doctoral thesis, TDX (Tesis Doctorals en Xarxa), 2022. http://hdl.handle.net/10803/673798.

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Our current knowledge about the function of human genes is mostly based on data from genetic studies using animal models. However, divergence among species may hamper the understanding of genetic mechanisms underlying human specific traits. Nowadays, an alternative to animal models stands on the generation of organ-like cultures derived from human pluripotent stem cells (hPSCs), the so called organoids. In the last years, the organoids have proved to recapitulate, in a high extent, the development, multicellular architecture, and physiology of human organs. To perform genetic studies in these valuable models, methods for rapid and controllable genetic manipulation of hPSCs are needed. To fulfill this need, the generation of an inducible CRISPR platform (iCRISPR) previously enabled efficient and inducible genome editing in hPSCs. Nevertheless, iCRISPR had important limitations hampering further transgenesis for expanding its possible applications. Based on iCRISPR, this thesis describes the generation of a new iCRISPR2 (iC2) platform with improved versatility for transgenesis. We generated selection-free hPSCs with monoallelic insertions of an inducible Cas9 expression module at the AAVS1 locus. Engineered iC2 hPSCs were then exploited for the high-throughput generation of stable and inducible KO hPSCs lines, precise KI hPSCs lines, and a reporter hPSCs line. In addition, the iC2 platform was repurposed for the generation of hPSCs for controlled gene regulation by CRISPR/dCas9 systems. Finally, some stable and inducible KO hPSCs were differentiated into kidney organoids to study the role of LHX1 and VHL in kidney development and disease. Our results showed that LHX1 function is required for renal vesicle formation prior nephrogenesis, as well as that VHL depletion impairs mitochondrial respiration in tubular cells derived from kidney organoids. On balance, iCRISPR2 technology could be applied in any hPSCs-based differentiation model to systematically dissect gene function in human development, physiology and disease Nuestros conocimientos sobre la genética humana se basan mayormente en datos procedentes de estudios genéticos realizados con modelos animales. Sin embargo, la divergencia entre especies puede dificultar la comprensión de los mecanismos genéticos que subyacen a los rasgos específicos del ser humano. Actualmente, una alternativa al uso de modelos animales se basa en la generación de cultivos similares a órganos mediante la diferenciación de células madre pluripotentes humanas (hPSCs), llamados organoides. En los últimos años, los organoides han demostrado recapitular, en gran medida, el desarrollo, la arquitectura multicelular y la fisiología de los órganos humanos. Para realizar estudios genéticos en estos valiosos modelos, se necesitan métodos de manipulación genética rápida y controlable de las hPSCs. Para cubrir esta necesidad, la generación previa de una plataforma inducible de la tecnología CRISPR (iCRISPR) ha permitido la edición del genoma de las hPSCs de una forma eficiente y regulable. Sin embargo, iCRISPR tiene importantes limitaciones que dificultan procesos de transgénesis que permitan ampliar sus posibles aplicaciones. Basada en iCRISPR, esta tesis describe la generación de una nueva plataforma iCRISPR2 (iC2), con mayor versatilidad para la transgénesis. En este trabajo hemos generado hPSCs libres de genes de resistencia a antibióticos y con inserciones monoalélicas en el locus AAVS1 que permiten la expresión inducible de la proteína Cas9. La nueva plataforma iC2 ha sido explotada para modificar con un alto rendimiento el genoma de hPSCs e introducir así mutaciones de pérdida de función génica (KO) estables e inducibles, mutaciones precisas y genes reporteros. Además, la plataforma iC2 se ha re propuesto para la generación de hPSCs que permitan la regulación controlada de genes mediante sistemas CRISPR/dCas9. Finalmente, algunas hPSCs con mutaciones KO se han diferenciado en organoides renales para poder estudiar el papel de genes como LHX1 o VHL en el desarrollo y la enfermedad renal. Nuestros resultados han demostrado que la función de LHX1 es necesaria para la formación de vesículas renales antes de la nefrogénesis, así como que la pérdida de VHL perjudica la respiración mitocondrial en las células tubulares derivadas de organoides renales. En definitiva, la tecnología iCRISPR2 podría aplicarse en cualquier modelo de diferenciación basado en hPSCs para diseccionar sistemáticamente la función de los genes en el desarrollo, la fisiología y las enfermedades humanas.

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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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44

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.

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Pröbsting, Michael Verfasser], Daguang [Akademischer Betreuer] [Cai, and Christian [Gutachter] Jung. "Application of CRISPR-Cas9 genome editing systems for improving oilseed rape (Brassica napus) disease resistance against Verticillium longisporum / Michael Pröbsting ; Gutachter: Christian Jung ; Betreuer: Daguang Cai." Kiel : Universitätsbibliothek Kiel, 2020. http://nbn-resolving.de/urn:nbn:de:gbv:8-mods-2020-00084-7.

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46

Pröbsting, Michael [Verfasser], Daguang [Akademischer Betreuer] Cai, and Christian [Gutachter] Jung. "Application of CRISPR-Cas9 genome editing systems for improving oilseed rape (Brassica napus) disease resistance against Verticillium longisporum / Michael Pröbsting ; Gutachter: Christian Jung ; Betreuer: Daguang Cai." Kiel : Universitätsbibliothek Kiel, 2020. http://d-nb.info/120658887X/34.

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47

Carayon, Alexandre. "Mise en place de l'identité musculaire durant la myogenèse embryonnaire chez la drosophile." Thesis, Toulouse 3, 2018. http://www.theses.fr/2018TOU30107.

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La diversité morphologique des muscles squelettiques permet la précision et la coordination des mouvements propres à chaque espèce animale. L'établissement du patron musculaire a lieu au cours du développement embryonnaire durant le processus de myogenèse. Il a été décomposé en quatre étapes chez la drosophile : la spécification de groupes de myoblastes équivalents (groupes promusculaires) à des positions précises du mésoderme, la sélection d'une ou plusieurs cellules progéniteurs à partir de chaque groupe, la division asymétrique des progéniteurs en cellules fondatrices des muscles, et enfin, la fusion d'une cellule fondatrice avec un nombre défini de myoblastes compétents pour la fusion qui forme une myofibre syncytiale. Ce processus aboutit à la mise en place d'un patron stéréotypé de muscles morphologiquement distincts par leur taille, orientation, forme, et sites d'attachement au squelette ; ces caractères définissant l'identité du muscle. Chez la drosophile, chacun des 30 muscles par hémisegment de la larve est constitué d'une seule myofibre. Il a été proposé que l'identité morphologique de cette fibre soit contrôlée par une combinatoire de facteurs de transcription identitaires (FTi) exprimés par la cellule fondatrice. Mon projet de thèse a porté sur le contrôle transcriptionnel de l'identité musculaire, avec comme modèle d'étude, un muscle dorso-latéral de la larve de drosophile, le muscle DA3 dont un FTi est Collier/EBF (Col). La transcription de col est activée dans un groupe promusculaire, puis transitoirement dans les quatre progéniteurs issus de ce groupe, avant d'être maintenue spécifiquement dans la myofibre DA3. Dans des embryons mutants pour col, le DA3 est transformé en muscle plus dorsal, DA2. Les travaux précédents de l'équipe ont montré que la transcription de col dans le lignage DA3 est contrôlée par deux modules cis-régulateurs, EarlyCRM et LateCRM, séparés physiquement sur le chromosome et agissant séquentiellement. Leur chevauchement temporel d'activité restreint au progéniteur DA3 et l'autorégulation directe du LateCRM ont mené à l'hypothèse d'un mécanisme de " passage de témoin " entre ces deux CRM, spécifique au progéniteur DA3. L'objectif de ma thèse était de tester cette hypothèse et de comprendre comment une information temporelle et spatiale intégrée par un CRM est transmise à un autre CRM, pour définir une identité cellulaire, une question fondamentale au-delà du cas d'espèce que constitue le muscle DA3.[...] The morphological diversity of skeletal muscles allows the precision and coordination of movements specific to each animal species. Establishment of a stereotypic pattern of muscles takes places during the process of myogenesis. Studies in Drosophila, an insect model, have identified four steps in this process: the specification of equivalence groups of myoblasts (promuscular clusters) at defined positions within the somatic mesoderm, the selection of progenitor(s) from each group, asymmetric division of each progenitor into post-mitotic muscle founder cells, and finally the fusion of each founder cell with a given number of fusion competent cells to form a syncytial myofiber. This dynamic, integrated process leads to establishing a stereotyped pattern of morphologically distinct muscles which can each be distinguished, based on size, orientation, shape, sites of attachment to the skeleton, all properties defining muscle identity. In the Drosophila larva, each of the about 30 different muscles per hemisegment is made of a single myofiber. It has been proposed that final morphology of a myofiber reflects the combinatorial code of identity Transcription Factors (iTF) expressed by its founder cell, although many questions remain unanswered. My thesis project aimed at better understanding the mechanism of specification of muscle identity, using as model a dorso-lateral muscle of the Drosophila larva, the DA3 muscle whose identity is controlled by the Collier/EBF (Col) iTF. col transcription is activated in one promuscular cluster, transient in the 4 progenitors issued from this cluster and stably maintained in the DA3 myofiber. In col mutant embryos, the DA3 muscle is transformed into a more dorsal, DA2-like muscle. Previous work has shown that col transcription in the DA3 lineage is controlled by two cis-regulatory modules (EarlyCRM and LateCRM), physically distant on the chromosome and acting sequentially. The temporal overlap of EarlyCRM and LateCRM in the DA3 progenitor and direct col autoregulation via the LateCRM led to hypothesize a handover between the two CRM in the DA3 progenitor. One goal of my thesis project was to challenge this hypothesis and understand how positional and temporal information integrated by EarlyCRM could be memorized via LateCRM, in order to specify cell identity, a fundamental question of developmental biology beyond the specific case of the Drosophila DA3 muscle. [...]

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Youssef, Divana. "Recherches de méthodes innovantes issues des biotechnologies pour l'amélioration génétique du blé tendre (Triticum aestivum L.)." Thesis, Université Clermont Auvergne‎ (2017-2020), 2017. http://www.theses.fr/2017CLFAC054.

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L’amélioration génétique du blé tendre (Triticum aestivum L.), une des trois céréales les plus cultivées, représente un intérêt stratégique pour la sécurité alimentaire de la population mondiale. Cette amélioration génétique va nécessiter une meilleure compréhension des mécanismes moléculaires et physiologiques mis en jeu, et va aussi réclamer une efficacité accrue dans notre capacité à intervenir finement sur le génome. Les avancées majeures réalisées dans le domaine des biotechnologies ces dernières années permettent d’envisager de nouveaux champs d’action pour appréhender le fonctionnement des caractères d’intérêt agronomique du blé tendre, ainsi que pour son amélioration génétique, et fournissent également de nouveaux outils pour innover dans le domaine de l’édition des génomes. Nous avons cherché dans le cadre de cette thèse à développer des innovations chez blé tendre à partir de trois nouveaux outils issus des biotechnologies. Nous avons tout d’abord montré que l’extinction du gène pds par une stratégie de micro ARN artificiel à partir d’un micro ARN de riz permettait d’obtenir le phénotype attendu, et que l’expression du micro ARN artificiel était reliée à ce phénotype. Nous avons commencé à explorer la possibilité d’utiliser des microARN de blé pour réaliser la même extinction, sans résultat pour l’instant. Nous avons ensuite montré que des coupures spécifiques d’une séquence donnée peuvent être obtenues in vivo chez le blé tendre à l’aide d’une méganucléase, et que lorsque les sites de coupure encadrent une séquence donnée, une délétion du fragment encadré peut être obtenue. Nous avons enfin réalisé les premiers essais du système CRISPR-Cas9 au laboratoire et généré une lignée exprimant le transgène Cas9 de façon constitutive. Des résultats inattendus obtenus dans le cadre de ces expérimentations nous ont de plus permis d’améliorer le procédé de transformation génétique du blé tendre utilisé au laboratoire. Les applications de nos résultats pourront être utilisées pour des expérimentations de validation de gènes et de compréhension des mécanismes moléculaires associés, mais aussi à l’avenir pour intervenir directement et de plus en plus finement sur le génome du blé. Les choix stratégiques en termes de développement technologique et d’innovation dans le domaine des biotechnologies et dans le cadre des objectifs d’un laboratoire public sont discutés The genetic improvement of common wheat (Triticum aestivum L.), one of the three most cultivated cereals, is of strategic interest to the food security of the world's population. This genetic improvement will require a better understanding of the molecular and physiological mechanisms involved, and will also require increased efficiency in our ability to modify finely the genome. In recent years, the major advances in biotechnology have made it possible to envisage new fields of action for a deeply understanding of agronomic traits of wheat as well as for genetic improvement, and also provide new tools for innovate in the field of genome editing. In this PhD manuscript, we sought to develop innovations for wheat improvement using three new biotechnology tools. We first demonstrated that the extinction of the pds gene by a strategy of artificial micro RNA succeeded in the obtaining of the expected phenotype and that the expression of the artificial RNA was related to this phenotype. We have begun to explore the possibility of using wheat microRNAs to achieve the same extinction, with no results at this time. We have then shown that specific cuts of a given sequence can be obtained in vivo in wheat using a meganuclease, and that when the cleavage sites frame a given sequence a deletion of the framed fragment may be obtained. We finally carried out the first tests of the CRISPR-Cas9 system in the laboratory and generated a line expressing the Cas9 transgene constitutively. Unexpected results obtained during these experiments have also made it possible to improve the process of genetic transformation of soft wheat used in the laboratory. The applications of our results can be used for gene validation experiments and a better understanding of the molecular mechanisms involved, but also in the future for wheat genome editing. Strategic choices in terms of technological development and innovation in the field of biotechnology and within the framework of the objectives of a public laboratory are discussed

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Stens, Cassandra, Isabella Enoksson, and Sara Berggren. "The CRISPR-Cas system." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-171997.

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Derived from and inspired by the adaptive immune system of bacteria, CRISPR has gone from basic biology knowledge to a revolutionizing biotechnological tool, applicable in many research areas such as medicine, industry and agriculture. The full mechanism of CRISPR-Cas9 was first published in 2012 and various CRISPR-Cas systems have already passed the first stages of clinical trials as new gene therapies. The immense research has resulted in continuously growing knowledge of CRISPR systems and the technique seems to have the potential to greatly impact all life on our planet. Therefore, this literature study aims to thoroughly describe the CRISPR-Cas system, and further suggest an undergraduate laboratory exercise involving gene editing with the CRISPR-Cas9 tool. In this paper, we describe the fundamental technical background of the CRISPR-Cas system, especially emphasizing the most studied CRISPR-Cas9 system, its development and applications areas, as well as highlighting its current limitations and ethical concerns. The history of genetic engineering and the discovery of the CRISPR system is also described, along with a comparison with other established gene editing techniques. This study concludes that a deeper knowledge about CRISPR is important and required since the technique is applicable in many research areas. A laboratory exercise will not only inspire but also provide extended theoretical and practical knowledge for undergraduate students.

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Rabai, Aymen. "Correction de l'ADN in vitro et in vivo comme thérapie personnalisée pour les myopathies congénitales." Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ117.

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L’édition du génome utilisant CRISPR/Cas9 est récemment apparue comme une stratégie thérapeutique potentielle des maladies génétiques. Pour les mutations dominantes de type gain de fonction, la correction allèle-spécifique pourrait être l'approche la plus appropriée. Ici, nous avons testé l'inactivation ou la correction d'une mutation hétérozygote du gène de la dynamine 2 (DNM2) causant la forme autosomique dominante de la myopathie centronucléaire (CNM). Des ARN-guides tronqués ciblant spécifiquement l'allèle muté ont été testés sur des cellules de patients et des myoblastes d'un modèle murin. L'allèle muté a été ciblé avec succès et des clones ont été obtenus avec inactivation ou correction précise du génome. Les myoblastes Dnm2R465W/+ ont montré une altération de l'endocytose et de l'autophagie. L'inactivation ou la correction allèle-spécifique a normalisé ces phénotypes. L'allèle muté a également été ciblé avec succès dans les muscles de la souris Dnm2R465W/+. Ces résultats illustrent le potentiel de CRISPR/Cas9 à cibler et corriger de manière allèle-spécifique les mutations ponctuelles hétérozygotes de type de gain de fonction Genome editing with the CRISPR/Cas9 technology has emerged recently as a potential strategy for therapy in genetic diseases. For dominant mutations linked to gain-of-function effects, allele-specific correction may be the most suitable approach. Here we tested allele-specific inactivation or correction of a heterozygous mutation in the Dynamin 2 (DNM2) gene causing the autosomal dominant form of centronuclear myopathies (CNM). Truncated single guide RNAs targeting specifically the mutated allele were tested on cells derived from a mouse model and patients. The mutated allele was successfully targeted in patient fibroblasts and Dnm2R465W/+ mouse myoblasts, and clones were obtained with both precise genome correction or inactivation. Dnm2R465W/+ myoblasts showed an alteration in transferrin uptake and autophagy. Specific inactivation or correction of the mutated allele rescued these phenotypes. The mutated allele was also successfully targeted in Dnm2R465W/+ mouse muscles. These findings illustrate the potential of CRISPR/Cas9 to target and correct heterozygous point mutations leading to a gain-of-function effect in an allele-specific manner

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Дисертації з теми "CRISPR, Cas9, genome editing, gRNA"

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