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Determining the mechanism of off-target mutagenesis caused by CRISPR-Cas9 genome editing

Dobbs, Felix 2021. Determining the mechanism of off-target mutagenesis caused by CRISPR-Cas9 genome editing. PhD Thesis, Cardiff University.
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Genome editing using CRISPR-Cas9 holds considerable promise for the generation of cell and gene therapies that can treat human disease. Despite this, the safety of CRISPR-Cas9 has been questioned because it can induce off-target DNA double strand breaks (DSB) in the genome, which often lead to mutagenic outcomes that directly drive oncogenesis. Several next generation sequencing (NGS) approaches have been developed to address this, but are biased by the PCR-based NGS library preparation that they employ. Consequently, these approaches do not provide an accurate measurement of CRISPR-Cas9 off-target activity in the genome. This thesis describes a method, INDUCE-seq, that was developed to solve this problem. In Chapter III, the development, and characteristics of INDUCE-seq are shown. INDUCE-seq combines a PCR-free methodology with a novel use of the Illumina sequencing flow cell for DSB enrichment to eliminate amplification bias. Each INDUCE-seq read is equivalent to a single DSB-end, generating an undistorted, digital readout for genomic breaks in cells. Chapter IV demonstrates the application of INDUCE-seq for measuring induced and endogenous DSBs in live cells. This work reveals the characteristics of AsiSI-induced cleavage and benchmarks INDUCE-seq against alternative methods. Furthermore, distinctive non-random endogenous break distributions are shown for different cell types. In Chapter V, INDUCE-seq is applied to the study of CRISPR off-targets in the genome. INDUCE-seq demonstrates enhanced sensitivity, discovering novel CRISPR off-targets in live cells, in addition to known sites that were previously only detectable using in vitro approaches. These off-targets are further analysed in Chapter VI, where it is shown that the sensitivity of DSB detection by INDUCE-seq outperforms amplicon sequencing for measuring the frequency of mutational editing outcomes. In conclusion, this thesis presents the novel method INDUCE-seq for measuring genomic DSBs. The information provided by INDUCE-seq will directly enable the development of safer CRISPR-based cell and gene therapies.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Medicine
Date of First Compliant Deposit: 13 August 2021
Last Modified: 13 Aug 2022 01:30

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