Elucidating the mechanism of topology-induced CRISPR/Cas9 off-target activity
File(s)
Author(s)
Smith, Quentin
Type
Thesis
Abstract
Bacteria developed CRISPR as an immune response to bacteriophage infections. Over the past few decades, it has then been harnessed as molecular scissors to edit genomes and cure genetic diseases. Recent approval by the MRHA and FDA to treat blood related diseases reveals the potential of curing many additional genetic diseases. However, Cas9 is not perfect, as it can cause unintended off-target editing genome-wide. These genome- wide off-targets pose a great barrier for the safety of using CRISPR/Cas9 in therapeutic applications. Most recent work from the Rueda lab and others have demonstrated that Cas9 off-target activity is modulated by DNA topology. In my thesis, I take multidisciplinary approaches to understand DNA topology and its influence on Cas9 targeting. I first developed small programmable DNA minicircles that can be negatively supercoiled ((-)SC) and showcase its many applications. Next, I use these (-)SC DNA minicircles to interrogate Cas9 targeting, I performed CryoEM to capture dCas9 and WT Cas9 bound to on- and off- target substrates. The series of structures reveal large conformational changes from previous structures on linear substrates, new mismatch geometries and potential new contacts that have been observed when Cas9 encounters (-)SC substrates. In addition, single molecule correlated optical tweezers and fluorescence experiments showcase HNH conformational dynamics that corroborate structural findings of Cas9 encountering negatively supercoiled DNA. Finally, I have undertaken protein engineering to develop the next-generation of high-fidelity Cas9 variants. Given that (-)SC induces Cas9 off-target activity, I have fused Cas9 with type I DNA topoisomerases, with the hypothesis through modulating local DNA topology, off-target Cas9 will be evicted. Taken together, the findings presented in this thesis are important for the development of next-generation high-fidelity Cas9 variants that reduce off-target activity.
Version
Open Access
Date Issued
2024-12-11
Date Awarded
2025-06-01
Copyright Statement
Attribution-NonCommercial 4.0 International Licence (CC BY-NC)
License URL
Advisor
Rueda, David
Publisher Department
Department of Medicine
Department of Infectious Disease
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)