Global ETD Search
Search theses and dissertations gathered from participating repositories worldwide. Every result links back to the library that holds it. No account is needed.
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Showing 1 to 20 of 30 for “"dCas9"”.
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Competition-based CRISPR-dCas9 transcriptional control mechanisms and application of dCas9 biosensors for high-throughput, cell-based protease inhibitor screens
Catalytically-dead Cas9 (dCas9) is a programmable transcription factor that can be targeted to promoters through the design of small guide RNAs (sgRNAs), where it can function as an activator or repressor. In Chapter 1 of this thesis, I outline the multitude of tools and applications that have been …
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CRISPR-based Transcriptional Activators for Dissecting Human Gene-Regulatory Mechanisms
… incompletely characterized. Here we expanded the dCas9-based activator toolbox to incorporate human CBP and GCN5 and tested their efficacy as transcriptional activators. For the optimization of the dCas9-toolbox, we showed that affinity-purification-mass spectrometry could be applied to identify …
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Resource competition in CRISPR-mediated gene regulation
… due to its modular nature: the same dCas9 effector can target different genes simply by changing the associated gRNA. However, multiplexing requires the sharing of limited amounts of dCas9 protein among multiple gRNAs, leading to resource competition. In turn, competition between …
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Epigenetic and Ubiquitin-Proteasome Mechanisms of Obesity Development
… with quantitative molecular assays and CRISPR-dCas9 manipulations to test the role of hypothalamic 1) DNA 5-hmC levels, 2) Pomc methylation, and 3) dysregulated ubiquitin-proteasome signaling in abnormal weight gain following exposure to obesogenic diets. We found that males, but not females, …
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Development of CRISPR/Cas9-based gene drive biotechnology in S. cerevisiae
… variant that has lost its nuclease activity (dCas9). We developed a CRISPR/dCas9 system by tagging dCas9 with transcriptional regulators. Our experiments demonstrated that CRISPR/dCas9 could activate target gene transcription when tagged with the transcriptional activator VPR and repress gene …
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Exploring Non-transgenic CRISPR-Cas9 gRNA Delivery Using Transactivation in Nicotiana benthamiana
… in a complex with gRNA. This deactivated Cas9 (dCas9) can be fused with transcriptional activators or repressors to adjust the expression of a target gene. While CRISPR-Cas9 is already widely used in plants, there are limitations to the system’s delivery and efficiency. The aims of the research …
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CPISPR-CAS: From a Prokaryotic Immune System to a Gene Editing Tool
… of Cas9, creating a catalytically dead Cas9 (dCas9) which retained DNA binding activity. We demonstrated that dCas9, when programmed with appropriate crRNAs, acted as a transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription …
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Development and application of synthetic biology tools for natural product research
… interspaced short palindromic repeats (CRIPSR)-dCas9 system for gene repression in both Streptomyces lividans 66 and Streptomyces albus J1074. By fusing the RNA polymerase omega factor (RNAP-ω) to dCas9, it was able to activate the silent undecylprodigiosin (RED) gene cluster in S. lividans 66. …
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The Use of Catalytically Dead Cas9 to Identify Key Transcription Regulators in Triple Negative Breast Cancer
… catalytically dead version of the Cas9 protein (dCas9). In particular, we focused on transcription regulators like FOXC1, NFIB and NFE2L3. We then performed RIME proteomics to identify which proteins are in close proximity to dCas9 and thus potentially bound to these putative regulatory regions. …
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Genetic Mechanisms of Transcriptional Regulation in Childhood Acute Lymphoblastic Leukemia
… with ALL and conducted high throughput CRISPR/dCas9 screening in an engineered ARID5B mCherry knock-in cell line. Effects of genetic polymorphism on binding affinity of transcription factor and chromatin accessibility were subsequently assessed. We applied CRISPR/dCas9 to investigate …
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Dynamic regulation of pathways by down-regulating competing enzymes
… by engineering the Streptococcus pasteurianus dCas9 and sgRNA. We demonstrate a robust and titratable gene depletion system, with up to 100-fold repression in [beta]-galactosidase activity in P. aeruginosa. We performed the first in vivo characterization of PAM site preferences of S. …
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Biomolecular and computational frameworks for genetic circuit design
… Second, we tested catalytically-inactive dCas9, which employs small guide RNAs (sgRNAs) to repress genetic loci via the programmability of RNA:DNA base pairing. To this end, we use dCas9 and synthetic sgRNAs to build transcriptional logic gates with high on-target repression and negligible …
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Epigenetic and post-transcriptional regulation of gene expression in pluripotent stem cells, differentiation and metastasis
… - protein fusions of RNA-guided DNA binding dCas9 - will enable experiments to directly test biological processes at defined genomic loci and expands the toolbox for synthetic biology and potentially opens up opportunities for novel therapeutics.
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The design, characterization, and application of CRISPR-based tool in human cells
… the transcriptional responses associated with dCas9-based histone acetylation. (2) construct a modular user-defined platform for the precise recruitment of proteins with diverse applications in targeted transcriptional activation, mammalian metabolic engineering, and identifying synergistic …
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Creation of Versatile Cloning Platforms for Transgene Expression and Epigenome Editing and Their Application to Pancreatic Islet Biology
… as a foundation, we also developed an S. aureus dCas9 epigenetic engineering platform, pMAGIC, that enables the packaging of 3 guide RNAs with Sa-dCas9 fused to one of five epigenetic modifiers into a single vector. Using pMAGIC-derived adenoviruses, we functionally validated the regulation of …
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Mammalian genome engineering: Fundamental investigation and method development
… studied the target-search mechanism of TALE and dCas9 proteins in live-mammalian cells using single molecule microscopy. The first part focuses on elucidating TALE search mechanism in vivo. For the first time, TALE genome-search has been visualized in real-time in live-cells and search kinetics …
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The role of Igf2 methylation and histone ubiquitination in age-related memory decline
… impacts memory of aged rats, we used a CRISPR dCas9-TET1 system to oxidate DNA methylation marks at the Igf2 promoter, converting this region to a 5-hydroxymethylation (5-hmC) state that increase gene transcription. Remarkably, this manipulation led to improved memory retention in aged, but not …
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An integrated CRISPR-Cas toolkit for engineering human cells
… dead version of the CRISPR/Cas protein Cas9 (dCas9) with mammalian transcriptional activator VP64 and mammalian transcriptional repressor KRAB, we build and characterize tunable, multifunctional and orthogonal CRISPR/Cas transcription factors (CRISPR-TFs) in human cells. By integrating …
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Acute Manipulation of Erna Level For Dissecting Its Roles In Transcriptional Regulation
… methods were tested on a testbed eRNA, including dCas9 based CRISPR activation or inhibition system and antisense oligonucleotide (ASO) based RNA inhibition system. I found that these CRISPR based systems show variable effects and sub-optimal acute perturbation of target eRNA, while ASOs are more …
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Investigating the transcriptional regulation of BCL11A in Triple-negative Breast Cancer
… the mutated catalytically inactive Cas9 (dCas9) to target these sites and to pull down proteins in close proximity. This novel approach led to the identification of DEK, SSRP1 and PSIP1 as potential regulators of BCL11A’s expression. Experimentally, I found knocking- down DEK, SSRP1 and …
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