aimofcontact:"Shortly after we decided to use prime editing as the gene editing method for our cystic fibrosis therapy, Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven and his colleagues published a paper directly related to our research[1]. In contrast to our approach, Bulcaen et al. 2024 targeted other, less common but drug-refractory CFTR-specific mutations (L227R- and N1303K). ",
insights:"The interview with Mattijs was valuable for us in a lot of ways. At that point in the project we were starting to design the components of our prime editor, but we were lacking a broader overview over the state of the field. Mattijs gave us this insight, mentioning techniques like PE3b systems, dsgRNAs and a talk given by David Liu,[Link] the principal investigator behind prime editing that helped us to consider further novel advancements in in Prime Editing and include them into our project. He discussed with us the difficulties that might await us when targeting the CFTR F508del deletion and mentioned that insertions of all the edits possible with prime editing are the hardest to make, the recognition of edits in the region might attract mismatch repair systems and the chromatin organization might negatively impact prime editing efficiency. Also, we learned a lot about how to design our pegRNAs, with important inputs being the 3’ stem loop motif trevopreQ1 used by Mattijs in his publication and the suggestion to use prediction tools to evaluate sgRNA spacer cutting efficiency. We reviewed our approach of testing pegRNAs using the PEAR reporter system and Mattjis recommended to use HEK cell lines for screening because of their easy handling and naturally impaired mismatch repair system. ",
implementation:"The inputs given by Mattijs directly impacted our design choices for multiple parts of the project. For the pegRNA design, we decided to use the same 3’ motif as Mattijs had used and also, like he suggested, checked our spacer candidates for predicted cleavage efficiency. Also we used HEK cells for screening our pegRNAs. We looked further into PE systems that influence cellular mismatch repair (such as PE4) and tried to include these into our design. ",
