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 PEmaxDRNaseH, the truncated editor used in dualAAV delivery systems. Finally, we generated Cas9 domains that improve prime editing. These resulting editors (PE6a-g) enhance therapeutically relevant editing in patient-derived fibroblasts and primary human T-cells. PE6 variants also enable longer insertions to be installed in vivo following dual-AAV delivery, achieving 40% loxP insertion in the cortex of the murine brain, a 24-fold improvement compared to previous state-of-the-art prime editors.

Automated summary

Prime editing enables precise genome edits in living cells, and protein evolution and engineering have been used to generate prime editors with improved efficiency and reduced size. Different types of reverse transcriptases specialize in different types of edits, and the new editors show enhanced therapeutic editing. The improvements in prime editing have significant implications for gene editing technologies.