Enhancing prime editing by fusing polymerase substrate-binding proteins to reverse transcriptase

Prime editing (PE) enables precise small DNA changes yet often shows modest efficiency in human cells. Recent advances indicate that the activity of the reverse transcriptase (RT) component is a key determinant of PE performance. Here, we adapt a principle from natural polymerases by fusing a polymerase substrate binding protein (PSBP) with RT to enhance PE. In a twelve-member screen, multiple PSBPs increased editing efficiency, with a compact HMG box module, NHP6A, emerging as a representative lead fusion. NHP6A fused to RT significantly increases intended edits across substitutions, insertions, and deletions while indels remain low. The enhancement is broadly compatible with a nicking sgRNA (PE3), transient mismatch repair inhibition via MLH1dn (PE4), and structured 3' pegRNA extensions (epegRNA). The PSBP module also synergizes with the La protein-assisted pegRNA tail stabilization (the PE7 system) for additional improvements. Using NHP6A together with La, we efficiently installed four clinically relevant alleles with high product purity. Finally, we validate that PSBP fusion can be consolidated into a single component prime editor without loss of activity. These results establish PSBP fusion as a precise route to improve prime editing outcomes and support integrating compact accessory modules into next-generation PE platforms.