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Durham e-Theses
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Functional characterization of CRISPR-Cas interactions with DNA

LOSITO, MARIALUCREZIA (2022) Functional characterization of CRISPR-Cas interactions with DNA. Doctoral thesis, Durham University.

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The recent Nobel-prize-winning CRISPR technology has revolutionised the gene-editing field with its ability to precisely modify any target DNA sequence and its relevance in a wide range of applications, from functional
characterisation of gene variants in biological sciences to mutation of plant genomes in agriculture. New prospectives for CRISPR-therapeutic applications are also rapidly advancing, particularly for the modification of disease-causing genes and ex-vivo editing of immune cells for cancer treatment. However, off-target editing exists as collateral damage and it represents a significant hurdle to realise CRISPR’s full potential. To date, the scientific community lacks extensive knowledge regarding the impact of the eukaryotic cellular context on Cas nucleases activity, and how this affects Cas efficiency and specificity on human DNA. In this thesis, I use a combination of cellular and single-molecule assays to investigate this specific topic. First, I developed a strategy to demonstrate that Cas9 specificity is diminished by a local distortion of the DNA 3D structure in human cells. Next, by using the CRISPR
system as a tool for gene expression regulation, I investigated the correlation between target transcription in cells and Cas9 editing efficiency. Together this first part of my work suggests that in vivo processes, which occur in eukaryotic cells and destabilise the DNA structure, have
the potential to induce off-targets. However, this effect is highly dependent on the target itself and its genomic context. Finally, I extended my study to two other CRISPR-Cas systems: Cas12a and a new engineered Cas (AZ-Cas9). By applying single-molecule technologies, I
contributed to the characterization of these nucleases, and obtained new information about the mechanisms underlining the DNA target search, the binding and cleavage kinetics and the off-target discrimination. These findings fill important knowledge gaps for future applications of these variants, and will be useful for the rational design of new high-fidelity nucleases.

Item Type:Thesis (Doctoral)
Award:Doctor of Philosophy
Faculty and Department:Faculty of Science > Chemistry, Department of
Thesis Date:2022
Copyright:Copyright of this thesis is held by the author
Deposited On:17 Nov 2022 13:04

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