UBC Theses and Dissertations
Safety of genome editing : development of a fluorescent model system to investigate reducing off-target genome edits by base editors Morin, Tessa
Gene editing is a potential treatment for genetic diseases, but current gene editing technologies are limited in their use and safety. CRISPR-Cas9 installs double stranded breaks in the genome which can be hard to fix, as the homologous-driven repair mechanism it uses has low efficiency. Newer gene editors have more specificity, such as CRISPR/Cas9-derived base editors, which enzymatically convert a target base to another. However, base editors are also capable of creating unwanted mutations, known as off-target edits, which can potentially lead to tumourgenesis. The difficulty of detecting these base changes has resulted in a poor understanding of factors involved in off-target edits. For my study, I successfully developed a human cell fluorescent model system using a mutated Green Fluorescent Protein (GFP) reporter gene with a premature stop codon. After successful base editing to correct the mutation, the cells are analyzed using flow cytometry to quantify the editing efficiency. To simulate guide RNA (gRNA)-dependent off-target editing, I designed multiple mismatched sgRNAs with intentional mismatches to the target GFP sequence. These mismatched sgRNAs mimic the true GFP sgRNA matching incorrectly to sequences similar to GFP in the genome. I tested the model with three adenosine base editors with different deaminases: the new base editor ABE8e, which is known for its promiscuous editing activity, an ABE8e containing the safety mutation V106W to reduce off-target edits, and the standard ABE7.10. I hypothesized that using ABE8e V106W with off-target mismatched sgRNAs would have reduced genome editing compared to ABE8e. iv Using this model system, ABE8e was shown to correct GFP at 25.5%, ABE8e V106W at 28%, and ABE7.10 at 34%. ABE8e and ABE8e V106W showed no editing difference between the mismatched and matched sgRNAs, but ABE7.10 could not tolerate the mismatched sgRNAs. Sanger sequencing showed two bystander edits with ABE8e and ABE8e V106W, potentially reducing fluorescence of GFP. These results show the model is capable of modelling sgRNA-dependent off-target edits and was able to detect differences between versions of base editors. This mismatched model could be used to investigate off-target edits by base editors to help develop safe, effective gene therapies for treating genetic diseases.
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