Abstract
Genome editing can potentially be used to address a wide variety of genetic diseases. CRISPR-Cas9 can target a diseased gene and cut the DNA to activate the host DNA repair processes, which compete to repair the DNA cut. One of these processes, homology-directed repair (HDR), can particularly be used to
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introduce small mutations, which are able to correct genetic errors and cure genetic diseases. The issue is the competition with the non-homologous end-joining pathway (NHEJ), which often leads to small insertions or deletions at the cut site. In this work, we strived to improve the odds of HDR activation by several methods. We first applied a model from earlier work to measure the outcomes of DNA repair, based on the expression of fluorescent proteins. We then established a baseline of NHEJ and HDR activation by formulating lipid nanoparticles to deliver all of the necessary components for efficient CRISPR-Cas9 induced genome editing. This resulted in approximately 80% of cells undergoing the incorrect NHEJ repair pathway, and 20% the correct HDR pathway. Subsequently, we demonstrate that the balance between HDR and NHEJ is cell-cycle dependent as HDR becomes more active just before mitosis. We attempted to apply this by controlling the genomic localization of Cas9 in time, but were at this stage unable to improve the activation of HDR in this way without the use of toxic drug compounds. We then screened a library of oncological drugs to modulate the gene editing outcomes in an effort to find a therapeutic add-on to Cas9. We found that alisertib, an Aurora Kinase A inhbitor, was able to greatly increase the HDR outcome (>5 fold). We finally tinkered with the Cas9 structure to allow click-conjugation on its surface, to apply additional synergistic molecules with the complex formulation found earlier in the thesis. We were able to express azidophenylalanine on the Cas9 surface without loss of protein activity, and were succesful in conjugation of small interfering RNA which in this case targeted a model mRNA. Both the Cas9 and siRNA parts of the conjugate were active in model cells, paving the way for a Cas9-drug conjugate in the future. We finally reflect on the greater gene editing field which developed greatly in the years encompassing this work. We identify pros and cons of the use of CRISPR-Cas9 compared to base editing and prime editing, and that delivery of these complex molecules remains the biggest outstanding challenge of the field. We conclude that the results of this thesis contributed to a greater understanding of the mechanisms of therapeutic CRISPR-Cas9 induced gene correction, and especially on its pitfalls and complexity.
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