Prime editing is a CRISPR tool with great therapeutic potential. However, there are still many questions to be answered before it can be used routinely, including those related to its efficacy and mechanism. The aim of this work was to facilitate the application of prime editing by increasing its efficiency and expanding its targeting scope.
In the first part of the study, PEAR, a flexible fluorescent reporter system was developed to simplify the monitoring of prime editing efficiency in mammalian cells. Due to its versatility, PEAR can be used in multiple ways. Its sensitivity allows it to be used to compare the editing efficiencies of different prime editing setups (e.g. different pegRNA–second nicking sgRNA combinations), or to characterise the performance of different prime editors under the same editing conditions. PEAR experiments are generally less time- and cost-intensive than NGS-based approaches, so this can help to accelerate the development process of new prime editing systems. PEAR can also be applied as an enrichment marker to identify and select individual cells with high prime editing activity. PEAR can efficiently enrich all three main editing categories – insertions, deletions and substitutions – in multiple cell types. In 22 out of the 23 enrichment experiments performed, PEAR enrichment surpassed transfection marker enrichment, increasing the edited cell population by up to 84%.
The second part of the study contributed to the development of a new PE system, called proPE. ProPE can expand the targeting scope of prime editing by alleviating various bottlenecks that can reduce the efficiency of the original PE. This work assessed the effect of degraded RNAs on editing efficiency. It was shown using the PEAR system that degraded RNAs inhibit proPE to a lesser extent than PE. The efficiency enhancing effect of proPE on edits lying outside the target sequence was also evaluated exploring a new set of prime editing targets in the CYP1A1, CYP1A2 and CYP2B6 genes.
Together, the findings presented in the dissertation could help to promote the more widespread use of PEs by providing means to enhance editing efficiency and study PE variants.
