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.

中文翻译：

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通过核酸酶介导的引物编辑对水稻基因进行高效的原位表位标记


原位表位标记对于探测基因表达、蛋白质定位和自然细胞环境中蛋白质相互作用的动力学至关重要。然而，该技术在植物中的实际应用存在相当大的障碍。在这里，我们全面探讨了 CRISPR/Cas 核酸酶介导的引物编辑和不同的 DNA 修复途径在内源水稻 （Oryza sativa） 基因表位标记中的潜力。我们发现，与传统和其他衍生物 PE 方法相比，基于 SpCas9 核酸酶/微同源介导的末端连接 （MMEJ） 的引物编辑 （PE） 策略（称为 NM-PE）有助于更直接、更高效的基因标记。此外，基于 PAM 灵活的 SpRY 和 ScCas9 核酸酶的引物编辑器已被设计和实施，用于标记具有不同表位的内源基因，显着拓宽了 NM-PE 在水稻中的适用性。此外，NM-PE 已成功用于分别用 c-Myc 和 HA 标签同时标记水稻植株中的 MAP 激酶 （MPK） 基因 OsMPK1 和 OsMPK13。综上所述，我们的结果表明 NM-PE 工具包在水稻蛋白标记项目的靶向基因标记、基因功能研究和遗传改良方面具有巨大潜力。