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Efficient in situ epitope tagging of rice genes by nuclease-mediated prime editing

文献类型： 外文期刊

作者： Li, Xueqi 1 ; Zhang, Sujie 1 ; Wang, Chenyang 1 ; Ren, Bin 1 ; Yan, Fang 1 ; Li, Shaofang 4 ; Spetz, Carl 5 ; Huang, Jinguang 6 ; Zhou, Xueping 1 ; Zhou, Huanbin 1 ;

作者机构： 1.Chinese Acad Agr Sci, State Key Lab Biol Plant Dis & Insect Pests, Inst Plant Protect, Beijing 100193, Peoples R China

2.Chinese Acad Agr Sci, Natl Nanfan Res Inst, Key Lab Gene Editing Technol Hainan, Minist Agr & Rural Affairs, Sanya 572024, Peoples R China

3.Minist Agr & Rural Affairs, Sci Observing & Expt Stn Crop Pests Guilin, Guilin 541399, Peoples R China

4.Beijing Acad Agr & Forestry Sci, Natl Engn Res Ctr Vegetables, Beijing Vegetable Res Ctr, State Key Lab Vegetable Biobreeding, Beijing 100097, Peoples R China

5.Norwegian Inst Bioecon Res, Div Biotechnol & Plant Hlth, N-1432 As, Norway

6.Qingdao Agr Univ, Coll Plant Hlth & Med, Key Lab Integrated Crop Dis & Pest Management Shan, Qingdao 266109, Peoples R China

7.Zhejiang Univ, Inst Biotechnol, State Key Lab Rice Biol, Hangzhou 310058, Peoples R China

期刊名称：PLANT CELL （ 影响因子：11.6； 五年影响因子：12.1 ）

ISSN： 1040-4651

年卷期： 2024 年 37 卷 2 期

页码：

收录情况： SCI

摘要： 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. A straightforward and highly efficient derivative nuclease/microhomology-mediated end joining-based prime editing strategy enables single as well as dual gene tagging throughout the rice genome.

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