Mismatch prime editing (PE) gRNA (mpegRNA) increased PE efficiency and reduced indels

Prime editing systems enable accurate and efficient genome editing. However, its efficacy can be hindered by the propensity of pegRNA's 3' extension to form secondary structures due to high complementarity with the protospacer sequence. Additionally, prime editor's persistent presence can cause unintended indel formation over time, posing safety concerns in therapeutic applications. To address these issues, we developed a mismatched PE gRNA (mpegRNA) method that introduces mismatched bases to the protospacer sequence, reducing complementarity and secondary structure formation. As a result, mpegRNA was found to significantly enhance editing efficiency by up to 2.3 times(DNMT1,+3 A to T,PE2), Alternatively, the highest indel level can be reduced by 76.5% without compromising editing efficiency(HEK3, +1 T to A, PE3). Furthermore, we demonstrated that the mpegRNA strategy has a universal enhancement effect when combined with epegRNA or PE4/5max, highlighting its potential in both research and therapeutic applications. Overall, the mpegRNA strategy has the potential to improve prime editing efficacy and P/I ratio, and represents a promising development in genome editing technology.