Targeted insertion of regulatory elements enables translational enhancement in rice

Rundong Shen; Qi Yao; Dating Zhong; Xuening Zhang; Xinbo Li; Xuesong Cao; Chao Dong; Yifu Tian; Jian-Kang Zhu; Yuming Lu

doi:10.3389/fpls.2023.1134209

Targeted insertion of regulatory elements enables translational enhancement in rice

Front Plant Sci. 2023 Mar 31:14:1134209. doi: 10.3389/fpls.2023.1134209. eCollection 2023.

Authors

Rundong Shen^{1

2

3}, Qi Yao^{1

4}, Dating Zhong^{1

4}, Xuening Zhang^{1

4}, Xinbo Li^{1

2

3}, Xuesong Cao¹, Chao Dong^{2

3}, Yifu Tian^{1

2

3}, Jian-Kang Zhu^{2

5}, Yuming Lu^{1

4}

Affiliations

¹ Shanghai Center for Plant Stress Biology, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China.
² Center for Advanced Bioindustry Technologies, and Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.
³ Hainan Yazhou Bay Seed Lab, Sanya, Hainan, China.
⁴ Shanghai Collaborative Innovation Center of Agri-Seeds, Joint Center for Single Cell Biology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China.
⁵ Institute of Advanced Biotechnology, and School of Life Sciences, Southern University of Science and Technology, Shenzhen, China.

Abstract

In-locus editing of agronomically-important genes to optimize their spatiotemporal expression is becoming an important breeding approach. Compared to intensive studies on mRNA transcription, manipulating protein translation by genome editing has not been well exploited. Here, we found that precise knock-in of a regulating element into the 5'UTR of a target gene could efficiently increase its protein abundance in rice. We firstly screened a translational enhancer (AMVE) from alfalfa mosaic virus using protoplast-based luciferase assays with an 8.5-folds enhancement. Then the chemically modified donor of AMVE was synthesized and targeted inserted into the 5'UTRs of two genes (WRKY71 and SKC1) using CRISPR/Cas9. Following the in-locus AMVE knock-in, we observed up to a 2.8-fold increase in the amount of WRKY71 protein. Notably, editing of SKC1, a sodium transporter, significantly increased salt tolerance in T2 seedlings, indicating the expected regulation of AMVE knock-in. These data demonstrated the feasibility of such in-locus editing to enhance protein expression, providing a new approach to manipulating protein translation for crop breeding.

Keywords: breeding; enhancer; knock-in; rice; translational regulation.

Grants and funding

This study was supported by the National Natural Science Foundation of China (No. 32070396) and the National Key R&D Program of China (No. 2021YFD1201300).