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 37 for “"Sgrna"”.
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The Interrogation of Cas9 Aptamers and sgRNA Structures Through SELEX
… of Cas9, the interrogation and evolution of sgRNA scaffold, in addition to novel Cas9 binding RNAs, represent another echelon of development and therapeutic potential. Currently, the majority of research utilizes a singular guide RNA scaffold sequence (the sgRNA) for a given Cas protein …
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DESIGN AND INVESTIGATION OF DUMBBELL-SHAPED DNA MINIMAL VECTORS FOR CRISPR/CAS9 EDITING
… CRISPR/Cas9 gene editing tool. Our analyses of sgRNA structure revealed that unstructured CRISPR RNA (crRNA) domains in sgRNA exhibited higher target cleavage activities as compared with structured crRNA domains. We also found that molecular helper functions enhancing DNA nuclear import (SV40 …
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Identifying Epigenetic Regulators Of Tumor Dormancy And Recurrence
… accurate understanding of background levels of sgRNA skewing in an experimental system of interest, yet this type of characterization has not been performed for in vivo CRISPR screens in mouse models of cancer. To address this gap, we performed an in vivo CRISPR screen with a negative control …
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CRISPR activation screening at single-cell transcriptomic resolution: discovering new regulators of zygotic genome activation
… validate a method for detecting short guide RNA (sgRNA) molecules in single-cell RNA sequencing libraries. In chapter 4, I select 230 screen candidates and construct a lentiviral sgRNA library to target their promoters by CRISPRa. After transducing this library into mESCs and performing …
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Novel Lipid Polymer Design for Nucleic Acids Delivery
… reliable delivery systems for single-guide RNA (sgRNA)-Cas9 complexes. Lipid nanoparticles (LNPs) have emerged as one of the most successful non-viral carriers for nucleic acids, highlighted by the approval of the first small interfering RNA (siRNA) therapy and the rapid deployment of LNP-based …
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Development and validation of a CRISPR interference system for gene regulation in Campylobacter jejuni
… function. To achieve this, a “dead” cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81-176. The tool was validated through …
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Re-engineering of Dendrimer-Based Lipid Nanoparticles for Efficient and Precise HDR-Mediated Gene Editing
… nucleic acids necessary for HDR: Cas9 mRNA, sgRNA, and a donor DNA template containing the correct nucleic acid sequence, as well as the optimization of intra-particle nucleic acid ratios for efficient induction of HDR in vivo. To assess in vivo HDR efficiency, we employed xenograft tumors …
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Better safe than sorry: new CRISPR/Cas9 tools for improved genome engineering
… an expression unit for SpCas9, a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. This self-limiting circuit, by controlling Cas9 levels, results in increased genome editing specificity. For its in vivo utilization, we integrated SLiCES into a lentiviral delivery system …
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Development and application of a general method for somatic cell genetics
… using large-scale lentiviral single-guide RNA (sgRNA) pools and screened under both positive and negative selection. After the initial, proof-of-principle experiments, I constructed a genome-wide sgRNA library and screened for genes required for the proliferation and survival of the nearhaploid …
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In Situ Perturb-Seq of Transcriptomes and RNA Neural Recordings
… dataset that includes single guide RNAs (sgRNAs), neural activity, and transcriptomics. Targeted ExSeq, with the inclusion of no-gap padlock probes and SplintR ligase, can be applied to enhance the detection of sgRNA barcodes and targeted transcripts. In vitro and in vivo experimental …
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Optimization of molecular tools for high-throughput genetic screening
… high-throughput screening, both for shRNA and sgRNA based reagents. Using large shRNA efficacy datasets, we first designed an algorithm predicting the potency of shRNAs based on sequence determinants. Combined with a novel shRNA backbone that further improves the processing of synthetic shRNAs, …
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Investigating the role of sufT in Mycobacterium abscessus using CRISPRi
… the metal chelator deferoxamine. To this end, 4 sgRNAs were designed, two targeting MAB_2744c (sg2744F1, sg2744F2) and two targeting the upstream gene 2745c (sg2745F1 and sg2745F2) and used to generate 4 M. abscessus sufT knock-down strains (M. abscessus attB::pLJR962-2744c-a, M. abscessus …
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Genetic screens of RNA binding proteins identify a role for N6-methyladenosine in promoting B cell differentiation
… in lymphocytes. Initially, I generated custom sgRNA libraries targeting RBPs to facilitate CRISPR/Cas9 mediated gene knockout screens in either human or mouse contexts. Then, I employed these reagents to identify roles for RBPs in redox signalling, B cell lymphomagenesis, CD8 T cell activation, …
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A novel CRISPR-Cas9 platform with divergent targeting capabilities
… both predict the efficacy of single guide RNA (sgRNA) sequences targeting specific loci, as well as to classify and characterize the recently-discovered anti-CRISPR proteins.
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Characterisation of the Mechanism of Norovirus VPg-Nucleotidylylation
… I identified negative sense subgenomic RNA (-sgRNA), magnesium ions (Mg<sup>2+</sup>) and the viral protease (NS6) as essential components that facilitate specific and physiologically relevant VPg nucleotidylylation. Further, I showed precursor forms of NS6, NS56 and NS67, to stimulate the …
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Highly efficient Cas9 mediated transcriptional programming and delivery via Adeno-Associated Virus
… room for an additional promoter let alone an sgRNA expression cassette or accessory activation domains. Here, we detail a single-vector system for delivery of a miniaturized, potent Cas9 activator for AAV mediated transcriptional regulation in neuronal, hepatic, gonadal, and muscle derived …
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A Context-Forward In Vivo Functional Genomics Platform For Target Discovery and Establishing Vulnerability Context In Pancreatic Cancer
… intratumorally across PDX lines. Applying sgRNA direct-capture for targeted gene perturbation and sample multiplexing in scRNAseq, we validated PCEN-informed dependences (SMAD4, ZEB1, ILK) associated to the basal-like subtype intratumorally. Silencing these targets resulted in a significant …
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CRISPR/Cas9 screenings and in silico investigations nominate low-frequency alterations in DNA repair genes as biomarkers for castration-resistant prostate cancers.
… PCa cell lines and included the use of a custom sgRNA pooled library targeting 356 DRGs and the administration of several drugs, most of which are currently used in the clinic. To characterize the full spectrum of DRG aberrations, the in silico approach included the joint allele-specific level …
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Identification and characterisation of HNF1B as a pro-tumorigenic lineage factor in clear cell renal cell carcinoma
… genetic depletion screen in vitro with an sgRNA library targeting 50 TFs that are highly expressed in RCC. This highlighted HNF1B as a potential tumour dependency in ccRCC. I demonstrated that both HNF1B CRISPR knockout and CRISPRi knockdown conferred a strong selective disadvantage in …
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Interrogation and control of mammalian transcription
… of mediating robust upregulation with a single sgRNA (Konermann et al., Nature, 2015) which outperforms current systems by more than two orders of magnitude. I demonstrated that these transcriptional effectors are capable of activating up to 10 genes simultaneously, allowing for understanding of …
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