Addition of the T5 exonuclease increases the prime editing efficiency in plants

Prime editing (PE) is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates, but is limited by low editing efficiency. We previously fused the M-MLV reverse transcriptase to the Cas9 nickase, generating the PE2 (v1) system, but the editing efficiency of this system is still low. Here we develop different versions of PE2 by adding the 5'-to-3' exonuclease at different positions of the nCas9-M-MLV RT fusion protein. PE2 (v2), in which the T5 exonuclease fused to the N-terminus of the nCas9-MMLV fusion protein enhances prime editing efficiency of base substitutions, deletions, and insertions at several genomic sites by 1.7- to 2.9-fold in plant cells compared to PE2 (v1). The improved editing efficiency of PE2 (v2) is further confirmed by generating increased heritable prime edits in stable transgenic plants compared to the previously established PE-P1, PE-P2, and PPE systems. Using PE2 (v2), we generate herbicide-resistant rice by simultaneously introducing mutations causing amino acid substitutions at two target sites. The PE efficiency is further improved by combining PE2 (v2) and dualpegRNAs. Taken together, the increased genome editing efficiency of PE2 (v2) developed in this study may enhance the applications of PE in plants.Copyright & COPY; 2023, The Authors. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Automated summary

Prime editing (PE) is a versatile genome editing tool with low editing efficiency. This study developed different versions of PE2 by adding 5'-to-3' exonuclease at different positions of the nCas9-M-MLV RT fusion protein, resulting in PE2 (v2) which showed significantly improved editing efficiency in plant cells. The enhanced editing efficiency of PE2 (v2) was further confirmed in stable transgenic plants. The study also demonstrated the generation of herbicide-resistant rice using PE2 (v2) in combination with dualpegRNAs.