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Article|11 Dec 2023|OPEN
Efficient and heritable A-to-K base editing in rice and tomato 
Xinbo Li1,2 ,† , Jiyong Xie3,4 ,† , Chao Dong1,2 ,† , Zai Zheng1,2 , Rundong Shen1,2 , Xuesong Cao5 , Xiaoyan Chen1 , Mugui Wang1 , Jian-Kang Zhu1,5 , , Yifu Tian,1,2 ,
1Ministry of Agriculture and Rural Affairs Key Laboratory of Gene Editing Technologies (Hainan), Institute of Crop Sciences and National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya, Hainan 572024, China
2Hainan Yazhou Bay Seed Lab, Sanya, Hainan 572024, China
3Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 201602, China
4University of Chinese Academy of Sciences, Beijing 100049, China
5Institute of Advanced Biotechnology, and School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, China
*Corresponding author. E-mail: zhujk@sustech.edu.cn,tianyifu@caas.cn
Xinbo Li and Jiyong Xie,Chao Dong contributed equally to the study.

Horticulture Research 11,
Article number: uhad250 (2024)
doi: https://doi.org/10.1093/hr/uhad250
Views: 23

Received: 30 Aug 2023
Accepted: 15 Nov 2023
Published online: 11 Dec 2023

Abstract

Cytosine and adenosine base editors (CBE and ABE) have been widely used in plants, greatly accelerating gene function research and crop breeding. Current base editors can achieve efficient A-to-G and C-to-T/G/A editing. However, efficient and heritable A-to-Y (A-to-T/C) editing remains to be developed in plants. In this study, a series of A-to-K base editor (AKBE) systems were constructed for monocot and dicot plants. Furthermore, nSpCas9 was replaced with the PAM-less Cas9 variant (nSpRY) to expand the target range of the AKBEs. Analysis of 228 T0 rice plants and 121 T0 tomato plants edited using AKBEs at 18 endogenous loci revealed that, in addition to highly efficient A-to-G substitution (41.0% on average), the plant AKBEs can achieve A-to-T conversion with efficiencies of up to 25.9 and 10.5% in rice and tomato, respectively. Moreover, the rice-optimized AKBE generates A-to-C conversion in rice, with an average efficiency of 1.8%, revealing the significant value of plant-optimized AKBE in creating genetic diversity. Although most of the A-to-T and A-to-C edits were chimeric, desired editing types could be transmitted to the T1 offspring, similar to the edits generated by the traditional ABE8e. Besides, using AKBEs to target tyrosine (Y, TAT) or cysteine (C, TGT) achieved the introduction of an early stop codon (TAG/TAA/TGA) of target genes, demonstrating its potential use in gene disruption.