Method development for the generation and selection of precise genomic edits in human cells

Thesis

Gene editing is the process of generating targeted changes in the genome of an organism or cell. Changes can be imprecise insertions or deletions (indels) which generate null alleles and permit loss of function studies. Alternatively, an exogenous donor can be provided which allows precise changes to be copied into the genome. Precise editing can be used to advance scientific understanding by generating cell lines with specific mutations. Furthermore, precise editing has therapeutic potential: pathogenic mutations in patient’s cells can be reversed or compensatory mutations can be introduced.

The relatively low efficiency of precise editing limits applicability of genome editing and increases the burden of labour on the researcher. In this thesis, I describe my efforts to improve the efficiency of editing and enrich for correctly edited cells. I study the impact of published methods shown to improve efficiency of editing. I then develop two potential FACS-based methods to enrich for correctly edited cells. Firstly, I attempt to use molecular beacons to directly recognise sequence changes in mRNA, and find that, despite promising in vitro results, the beacons are highly non-specific within cells. I then develop a transient Cas9-based circuit to detect the presence of a genomically-integrated crRNA. This crRNA-expression system can be incorporated beside the intended edit, using an ssODN donor of less than 200 base pairs. Finally, I find that using the transient Cas9-based circuit, I can enrich for precisely edited HEK293T cells by 20-40 fold, achieving final editing efficiencies of 20-60% alleles.

Authors: Lightowlers, A

• DOI: 10.5287/ora-nzozvapdq
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English