Phage-assisted evolution and protein engineering yield compact, efficient prime editors

Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax. We discovered that different reverse transcriptases specialize in different types of edits and used this insight to generate reverse transcriptases that outperform PEmax and PEmaxΔRNaseH, the truncated editor used in dual-AAV delivery systems. Finally, we generated Cas9 domains that improve prime…

• NS
  National Science Foundation
• HH
  Howard Hughes Medical Institute
• BA
  Bill and Melinda Gates Foundation
• NI
  National Institutes of Health

  Awards: R01HL56067, R35GM118062, UG3AI150551, U01AI142756, RM1HG009490, R01EB027793