<p>For the prime editing of <strong>Cystic Fibrosis</strong> (CF), we on the one hand optimized a prime editing complex and on the other hand developed an efficient delivery system. For testing, we set up cell culture with model cell lines as well as primary cells taken from team members and a patient.</p>
<p>For editing, we first compared different existing prime editors <strong>(pCMV-PE2, pLV-PE_CO-Mini, pCMV-PE6c)</strong> and constructed a reporter plasmid simulating the <strong>CFTR context</strong>. In addition and to further enhance the editing process, we designed various pegRNAs tailored to our construct incorporating features such as <strong>silent edits</strong>, for a lower mismatch repair, and a 3′ stabilizing stem loop <strong>(tevropQ1)</strong>. The aim was to identify the most effective pegRNA for our specific target, which is why pegRNA especially for CFTR F508del mutation were designed.</p>
<p>For editing, we first compared different existing prime editors <strong>(pCMV-PE2, pMLV-PE_CO-Mini, pCMV-PE6c)</strong> and constructed a reporter plasmid simulating the <strong>CFTR context</strong>. In addition and to further enhance the editing process, we designed various pegRNAs tailored to our construct incorporating features such as <strong>silent edits</strong>, for a lower mismatch repair, and a 3′ stabilizing stem loop <strong>(tevopreQ1)</strong>. The aim was to identify the most effective pegRNA for our specific target, which is why pegRNA especially for CFTR F508del mutation were designed.</p>
<p>As proof of concept, we transfected these constructs in <strong>HEK293 and CFB41o- cells</strong> and observed significant prime editing of our reporter via fluorescence microscopy. We identified the PE6c editor and our pegRNA variant 4 as optimal. This resulted in our <strong>Best New Basic Part</strong>, <strong>PEAR_CFTR</strong>. Furthermore, we extended our approach to primary human nasal epithelial cells generated from our own nasal epithelial cells through nasal swabs. By cultivating them in Air Liquid Culture (ALI) and Apical-Our Organoids, we successfully tested our technologies <i>in vitro</i>, mimicking the <i>in vivo</i> situation.</p>
