Phage-assisted evolution and protein engineering yield compact, efficient prime editors
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This paper reports on the development of new optimized prime editors called PE6 variants (PE6a-g) that can improve prime editing outcomes in mammalian cells. The authors surveyed 59 different reverse transcriptase (RT) enzymes from diverse origins as potential prime editors. While 20 RTs showed some activity, most were far less efficient than the previously used M-MLV RT.
The authors used phage-assisted continuous evolution (PACE) to improve the activity of compact RTs from Tf1, Ec48, and GsI-IIC intron origins, increasing their mammalian prime editing efficiency up to 22-fold. Protein engineering was used to further optimize the Tf1 and Ec48 RTs, generating the 1.5 kb PE6b and 1.2 kb PE6a editors which can match the efficiency of the larger PEmax editor in cells and primary T cells.
For dual AAV delivery, the authors evolved and engineered highly processive M-MLV and Tf1 RTs, creating PE6c and PE6d. These variants recovered prime editing efficiency compared to the truncated PEmaxDRNaseH at challenging edits like long insertions. Evolved Cas9 domains were combined with PE6 RTs to create PE6e-g editors. Cas9 and RT domains can be mixed and matched to overcome deficiencies. In mouse brain, PE6c and PE6d increased the in vivo installation of long DNA sequences by up to 183-fold after dual AAV delivery compared to PEmaxDRNaseH.
https://www.cell.com/cell/pdf/S0092-8674(23)00854-1.pdf
