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 wide-ranging applications of prime editing, prime editors (PEs) have low editing efficiencies, especially in dicot plants. Therefore, PEs are barely used for genome engineering in dicot plant species. Here, based on previous approaches used to improve prime editing efficiency, we generated different combinations of PE components and prime editing guide RNAs (pegRNAs) and examined their prime editing efficiencies in Arabidopsis thaliana protoplasts as a dicot model system. We found that v4e2, in which PE was fused to viral nucleocapsid (NC) protein, RNase Hdeleted M-MLV RT, and a dominant negative version of human mutL homolog 1 (hMLH1dn), showed the highest prime editing efficiency in Arabidopsis protoplasts when it was co-transfected with dual enhanced pegRNA. Our results suggest that the v4e2 PE system could be used for efficient prime editing in dicot plants. [BMB Reports 2025; 58(2): 70-74].