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Title: Quantification of CRISPR-Cas9 diffusion dynamics in Escherichia coli
Author: Zaoui, Xavier Karim
ISNI:       0000 0004 9355 5928
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2020
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What do adaptive immunity, genetic engineering and antimicrobials have in common? CRISPR-Cas9, the popular enzymatic complex that produces DNA double-strand breaks when associated with a guide-RNA. Hundreds of labs routinely use this system to edit genomic DNA; however, some of the mechanisms by which it interacts with nucleic acid remain unclear. In my lab, we developed an expertise in the study of DNA recombination, DNA repair and DNA interactions in Escherichia coli. We use single-molecule fluorescent microscopy to collect images in real time, in vivo. During my PhD, I harnessed this expertise to follow the behaviour of the Cas9 protein under different conditions: various expression levels; various gRNAs; and various genomic targets. By observing the diffusion dynamics of the protein, I was able to quantify how different DNA interactions were impacting the motion of the protein in the cytoplasm and inferred that actual ON-target interactions were very rare throughout the lifetime of the protein. In contrast, the protein was mainly involved in non-specific OFF-target DNA interactions, in search of its actual target. Additionally, my results reveal the presence of a large fraction of non-specific interactions, hitherto not reported in the literature, owing to their absence of DNA modification. In total, this work offers a collection of highly quantitative measurements on the behaviour of a protein whose activity is central to many biologists, while shedding a new light on the importance of Cas9 searching and targeting mechanisms. Finally, it opens a discussion on the role of DNA recognition in the context of gene editing and antimicrobial resistance.
Supervisor: El Karoui, Meriem ; Pilizota, Teuta Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: CRISPR/Cas9 ; Escherichia coli ; gene editing ; fluorescence microscopy ; protein diffusion ; ON/OFF target