Phage-assisted continuous evolution and protein engineering yield compact, efficient prime editors and insights into prime editing
Prime editing (PE) is a versatile gene editing technology that enables a wide variety of precise genome edits in living cells without requiring double-strand DNA breaks or donor DNA templates. Despite several attempts to improve the reverse transcriptase (RT) domains of prime editors, PE systems predominantly use the engineered Moloney murine leukemia virus (M-MLV) RT from the original PE2 system, and the use of other RTs thus far has generally resulted in substantially lower prime editing efficiencies. Similarly, improving the engineered M-MLV RT and Cas9 domains to enhance editing properties beyond those of current-generation PEmax has also proven challenging. Here we use a combination of laboratory evolution and protein engineering to improve prime editing efficiencies and reduce prime editor size. We screened 59 natural RTs for prime editing performance and found that most exhibited minimal mammalian cell prime editing activity. Structure-guided engineering and phage-assisted continuous evolution (PACE) improved the mammalian cell prime editing activity of two novel, compact RTs by 7- to 22-fold on average compared to their wild-type counterparts, resulting in editing efficiencies comparable to those of PEmax but with 516-810 bp smaller gene sizes. We also discovered that highly evolved and engineered RTs specialize in different types of prime edits, and using this insight we generated RTs that outperform PEmax. RTs evolved with improved processivity enhance prime editing efficiencies for long, complex edits and twinPE (dual pegRNA) edits, while less processive RTs are optimal for small substitution edits. Finally, we used PACE to evolve Cas9 domains that can substantially improve prime editing. These findings reveal insights into the mechanism of prime editing and establish a suite of new prime editors (PE6a-6i) that offer size and efficiency advantages over PEmax for many different editing applications of therapeutic relevance, including in patient-derived fibroblasts and primary human T-cells. The PE6 RT variants and PE6 Cas9 variants can be used synergistically with each other, epegRNAs, the PEmax architecture, and mismatch repair evasion to yields cumulative benefits that define state-of-the-art PE systems.