Efficient in Situ Epitope Tagging of Rice Genes by Nuclease-mediated Prime Editing

In situ epitope tagging is crucial for probing gene expression, protein localization, and the dynamics of protein interactions within their natural cellular context. However, the practical application of this technique in plants presents considerable hurdles. Here, we comprehensively explored the potential of the CRISPR/Cas nuclease-mediated prime editing and different DNA repair pathways in epitope tagging of endogenous rice (Oryza sativa) genes. We found that a SpCas9 nuclease/microhomology-mediated end joining (MMEJ)-based prime editing (PE) strategy (termed NM-PE) facilitates more straightforward and efficient gene tagging compared to the conventional and other derivative PE methods. Furthermore, the PAM-flexible SpRY and ScCas9 nucleases-based prime editors have been engineered and implemented for the tagging of endogenous genes with diverse epitopes, significantly broadening the applicability of NM-PE in rice. Moreover, NM-PE has been successfully adopted in simultaneous tagging of the MAP kinase (MPK) genes OsMPK1 and OsMPK13 in rice plants with c-Myc and HA tags, respectively. Taken together, our results indicate great potential of the NM-PE toolkit in the targeted gene tagging for Rice Protein Tagging Project, gene function study and genetic improvement.