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

• DOI: 10.1016/j.cell.2023.07.039
• Publication Date: 2023-08-31T15:38:13Z
• Authors: Jordan L. Doman; Smriti Pandey; Monica E. Neugebauer; Meirui An; Jessie R. Davis; Peyton B. Randolph; Amber McElroy; Xin D. Gao; Aditya Raguram; Michelle F. Richter; Kelcee A. Everette; Samagya Banskota; Kathryn Tian; Y. Allen Tao; Jakub Tolar; Mark J. Osborn; David R. Liu
• Keywords: Resource; Cerebral Cortex; Mice; Humans; Animals; Brain; Bacteriophages; DNA-Directed RNA Polymerases; Protein Engineering

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 improved efficiency. Using phage-assisted evolution, efficiencies compact reverse transcriptases by up 22-fold generated that are 516–810 base pairs smaller than the current-generation editor PEmax. We discovered different specialize types used this insight outperform PEmax PEmaxΔRNaseH, truncated dual-AAV delivery systems. Finally, Cas9 domains improve editing. These resulting (PE6a-g) enhance therapeutically relevant patient-derived fibroblasts primary human T-cells. PE6 variants also enable longer insertions be installed vivo following delivery, achieving 40% loxP insertion cortex murine brain, 24-fold improvement compared previous state-of-the-art editors.