Insights into <scp>pegRNA</scp> design from editing of the cardiomyopathy‐associated phospholamban R14del mutation

Prime editing (PE) represents a transformative genome-editing technology and enables precise insertions, deletions, and base substitutions without introducing double-strand breaks, thereby reducing undesired indels and off-target effects. Despite advancements in enhanced prime editors and optimized prime editing guide RNAs (pegRNAs), designing effective pegRNAs remains a major challenge. The phospholamban (PLN) R14del mutation is associated with cardiomyopathies, making it a crucial target for precise gene-editing strategies. In this study, we explored pegRNA features that contribute to high editing efficiency using the FluoPEER.PLN R14del reporter cell line. Through systematic screening, we identified three pegRNAs with significantly enhanced editing efficiency. Our findings underscore the importance of pegRNA secondary structure and stability in optimizing prime editing, providing valuable insights into precise gene correction strategies.