THE SELECTION OF OPTIMAL pegRNAs TO ENHANCE THE EFFICIENCY OF PRIME EDITING IN AT-RICH GENOME REGIONS

Prime editing is a highly promising strategy for treating hereditary disorders due to its superior efficiency and safety profile compared to conventional CRISPR-Cas9 systems. This study is dedicated to development of a causal therapy for cystic fibrosis by targeting the F508del variant in the CFTR gene using prime editing, as this specific deletion accounts for a substantial proportion of cystic fibrosis cases. While prime editing has shown remarkable precision in introducing targeted genetic modifications, its application in AT-rich genomic regions, such as the one containing the F508del variant, remains challenging. To overcome this limitation, we systematically evaluated 24 pegRNAs designed for two distinct prime editing systems, PEmax and PE2-NG. The correction efficiency of the F508del variant reached 2.81% in basal lung cells from homozygous F508del patients, without normalization for transfection efficiency. However, the average transfection efficiency was only 11.9%, emphasizing the critical need for advancements in delivery methodologies. These findings highlight the potential of prime editing as a therapeutic approach for cystic fibrosis, while also underscoring the necessity for further optimization of both editing constructs and delivery vectors to achieve clinically relevant correction levels.