Development of a prime editor with improved editing efficiency in Arabidopsis.

Prime editing is widely used in many organisms to introduce site-specific sequence modifications, such as base substitutions, insertions, and deletions, in genomic DNA without generating double-strand breaks. Despite their wide-ranging applications, prime editors (PEs) have low editing efficiency, especially in dicot plants, and are therefore barely used for genome engineering in these plant species. Here, based on the previous approaches used to improve prime editing efficiency, we generated multiple different combinations of PE components and prime editing guide RNAs (pegRNAs) and examined their prime editing efficiency in Arabidopsis thaliana protoplasts as the dicot model system. We found that v4e2, in which PE was fused to the viral nucleocapsid (NC) protein, RNase H-deleted M-MLV RT, and a dominant negative version of human mutL homolog 1 (hMLH1dn), showed the highest prime editing efficiency in Arabidopsis protoplasts when co-transfected with dual enhanced pegRNA. Overall, our results suggest that the v4e2 PE system could be used for efficient prime editing in dicot plants.