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 20 for “"CRISPR interference"”.
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Development and validation of a CRISPR interference system for gene regulation in Campylobacter jejuni
… to address this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression and thereby investigate gene function. To achieve this, a “dead” cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. …
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Targeted depletion of RibF, a putative bifunctional FAD synthetase/ flavokinase in Mycobacterium smegmatis using CRISPR interference
… repression of downstream genes. CRISPR interference (CRISPRi) technology was used to generate an anhydrotetracycline (ATc)-inducible ribF hypomorph of M. smegmatis, a widely exploited mycobacterial model. Consistent with other organisms, ribF was shown to be essential for in vitro …
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Probing the Mycobacterial Cell Envelope: CRISPR interference-mediated investigation of two essential genes in the galactan biosynthetic pathway
… characterisation and cell morphology. The CRISPR interference (CRISPRi) genome editing system was used to target genes involved in galactan biosynthesis and generate transcriptional knock down of wecA (also known as rfe, MSMEG_4947) and rfbD (MSMEG_6369) in M. smegmatis, a widely exploited …
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CRISPRI-based high-throughput functional genomic approaches for use in mycobacteria
… viability. The recent optimisation of inducible CRISPR-interference for mycobacteria offers the potential to expand the high-throughput functional genomic toolkit. This thesis utilises CRISPR-interference for the development and validation of two high-throughput functional genomic approaches in …
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Deep representation learning for cancer transcriptomics: towards robust models for heterogeneous data
… observational patient data with interventional CRISPR-interference experiments to enhance survival prediction robustness. We find that deep learning holds great promise for addressing distributional shifts in cancer transcriptomics, but that much larger, more diverse datasets are needed, …
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Highly Saturated Transposon Sequencing identifies genes impacting Staphylococcus aureus pathogenesis in macrophages
… for optimal macrophage infection. We used CRISPR interference (CRISPRi) to functionally validate phenotypic contributions for a select subset of genes. Of the 20 genes passing validation, 7 had a previously identified role in S. aureus virulence, and 13 were newly implicated. Validated …
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Identification and characterisation of novel interactors of surfactant protein C
… validation of biologically relevant hits by CRISPR/Cas9 deletion of candidates involved in membrane trafficking, ubiquitination and lysosomal acidification. I found that many induced misprocessing and relocalisation of SFTPC-WT to the plasma membrane, phenocopying the I73T mutation. I …
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DEVELOPMENT AND APPLICATION OF CHEMOENZYMATIC TOOLS TO VISUALIZE MAMMALIAN PHOSPHOLIPASE D SIGNALING
… utility as a readout for a genome wide pooled CRISPR interference screen to identify regulators of protein kinase C (PKC)-dependent PLD signaling. Combining these two modern chemical tools has proven to be extremely powerful and allowed us to identify a new role for glycogen synthase kinase 3 …
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Genome-Scale Quantification of Target Vulnerability in Mycobacterium Tuberculosis
… To quantify gene vulnerability, we developed a CRISPR interference-based functional genomics method to systematically titrate the expression of nearly all essential genes and monitor fitness outcomes in the global pathogen, Mycobacterium tuberculosis (Mtb). Additionally we created an approach to …
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Disabling the intrinsic resistome of Mycobacterium tuberculosis: elucidating hierarchies of DNA repair and mutagenesis that undermine current antibiotic efficacy
… results from the Tn screen were validated using CRISPR interference targeting the identified genes. Of particular interest, we observed that UvrB was essential to compensate loss of DnaE2, whereas the reciprocal was less definitive: while DnaE2 appeared dispensable in MMC-treated uvrB, Tn …
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Investigating the role of sufT in Mycobacterium abscessus using CRISPRi
… routinely interspaced palindromic repeats interference (CRISPRi) system was recently optimized for transcription repression in mycobacteria. This study aimed to investigate the functionality of the mycobacterial CRISPRi in M. abscessus, specifically to investigate the role of the sufT …
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NEW FINDINGS INTO TARGET-DIRECTED MICRORNA DEGRADATION MECHANISM
… ligase substrate adapter. We repressed ZSWIM8 by CRISPR interference in a set of 8 different breast cancer (BC) cell lines, representing the entire spectrum of BC subtypes. Our analyses showed that loss of ZSWIM8 caused significantly increased accumulation of 31 miRNAs, without any evidence of …
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Creating effective drug discovery methods in Mycobacterium abscessus
… a phenotypic screening selection funnel, a CRISPRi arrayed library, and transcriptomic predictions of drug action. Increasing the focus of antibiotic discovery has been on narrow spectrum, species specific compounds, and I have focussed on developing tools in a single bacterium, …
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Whole-genome transposon mutagenesis to elucidate the genetic requirements for vitamin B12 biosynthesis and assimilation in mycobacteria
… targeted silencing of individual genes by CRISPR interference, candidate cobalamin biosynthesis genes were validated, providing functional evidence of their essentiality for metE survival in minimal medium, in turn supporting the validity of the cobalamin biosynthetic pathway constructed …
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A Chemical-Genetic Map of Drug Resistance in Mycobacterium Tuberculosis
… basis drug resistance in Mtb using a novel CRISPR interference (CRISPRi)-based chemical genetics platform. Our lab developed a CRISPRi library containing 100,000 unique knockdown strains and treated this library with a panel of some of the most important antitubercular drugs. The fitness of …
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Establishment of CRISPR-Cas-based gene deletions in Bacillus methanolicus and metabolic engineering towards the production of N-methylglutamate and putrescine
… advanced genome editing technologies, such as CRISPR-Cas systems, is key to improving this organism for industrial applications and increasing yields and productivity in line with sustainability goals. The first objectives in this study were i) to establish a CRISPR-Cas9-based gene deletion …
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Development of Improved Genetic Methods for Enterococcus Faecium
… regularly interspaced palindromic repeat (CRISPR)-Cas9 counterselection, I was able to produce both scar-less mutants via point mutations as well as controllable deletions of various sizes. Additionally, I report my system to have gene editing activity with PCR generated dsDNA templates. …
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Inquiries into the Mycobacterial Caseinolytic Protease System
… With the development of an inducible and tunable CRISPR interference (CRISPRi) platform in our laboratory, we were uniquely positioned to systematically investigate key questions in mycobacterial Clp biology, including which processes are regulated by the Clp system and contribute to its …
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Gene therapy for axon regeneration
… in cortical neurons by using vectors containing CRISPR-Cas9 and phosphoinositide 3-kinases. Chapter I is a literature review about cell surface receptors called integrins and their role in axonal regeneration. Integrins contribute to the spontaneous axon regeneration in the peripheral nervous …
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Investigating the Double-Edged Sword of Rifampicin Resistance in Mycobacterium Tuberculosis
… RifR Mtb in the clinic. Employing genome-wide CRISPR interference (CRISPRi) screens, we identified genes with differential sensitivity to inhibition in RifR Mycobacterium smegmatis (Msmeg; a model organism of Mtb) and Mtb, compared to a RifS strain. We identified genes involved in …