Fei Jidong, Zhao Dongdong, Pang Caiyi, Li Ju, Li Siwei, Qiao Wentao, Tan Juan, Bi Changhao, Zhang Xueli
College of Life Science, Tianjin Normal University, Tianjin, China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
Nat Commun. 2025 Jan 2;16(1):139. doi: 10.1038/s41467-024-55578-z.
Prime editing enables precise and efficient genome editing, but its efficacy is hindered by pegRNA's 3' extension, forming secondary structures due to high complementarity with the protospacer. The continuous presence of the prime editing system also leads to unintended indel formation, raising safety concerns for therapeutic applications. To address these challenges, we develop a mismatched pegRNA (mpegRNA) strategy that introduces mismatched bases into the pegRNA protospacer, reducing complementarity and secondary structure formation, and preventing sustained activity. Our findings show that mpegRNA enhances editing efficiency by up to 2.3 times and reduces indel levels by 76.5% without compromising performance. Combining mpegRNA with epegRNA further increases efficiency up to 14-fold, or 2.4-fold in PE4max/PE5max systems, underscoring its potential in research and therapy. AlphaFold 3 analysis suggests that the optimal mpegRNA structure contributes significantly to improved editing outcomes. Overall, mpegRNA advances prime editing technology, improving efficiency while reducing indels.
引导编辑能够实现精确且高效的基因组编辑，但其效率受到pegRNA 3'端延伸的阻碍，由于与原间隔序列高度互补而形成二级结构。引导编辑系统的持续存在还会导致意外的插入缺失形成，引发对治疗应用安全性的担忧。为应对这些挑战，我们开发了一种错配pegRNA（mpegRNA）策略，该策略在pegRNA原间隔序列中引入错配碱基，减少互补性和二级结构形成，并防止持续活性。我们的研究结果表明，mpegRNA可将编辑效率提高多达2.3倍，并将插入缺失水平降低76.5%，同时不影响性能。将mpegRNA与epegRNA结合可进一步将效率提高多达14倍，在PE4max/PE5max系统中提高2.4倍，凸显了其在研究和治疗中的潜力。AlphaFold 3分析表明，最佳的mpegRNA结构对改善编辑结果有显著贡献。总体而言，mpegRNA推动了引导编辑技术的发展，提高了效率同时减少了插入缺失。
