Yucai Li; Shaoya Li; Chenfei Li; Chen Zhang; Lei Yan; Jingying Li; Yubing He; Yan Guo; Yong Lin; Yangjun Zhang; Lanqin Xia.

Plant Communications, 2023, IF 10.50

DOI:10.1016/j.xplc.2023.100667

ABSTRACT:

Base editors (BEs) are promising tools that enable single-nucleotide substitutions in specific target genes, generating loss-of-function or gain-of-function mutations, which can greatly accelerate plant functional genomics and crop improvement (Ren et al., 2018; Zeng et al., 2020; Xu et al., 2021; Yan et al., 2021; Li et al., 2023). To date, three major classes of BEs have been engineered: cytosine BEs (CBEs) for C-to-T transitions (Komor et al., 2016), adenine BEs (ABEs) for A-to-G transitions (Gaudelli et al., 2017), and glycosylase BEs (CGBEs) for C-to-G transversions (Kurt et al., 2021; Zhao et al., 2021). ABE enabling A-to-K (K = G or T) editing remains to be exploited in both mammalian cells and plants. Recently, an adenine transversion BE (AYBE) was developed for A-to-Y (Y = C or T) transversion editing in human cells by fusing an ABE, ABE8e, with either the human hypoxanthine excision protein N-methylpurine DNA glycosylase (hMPG) or its mutant (mhMPG) (Tong et al., 2023) (Supplemental Figure 1). Development of a plant AYBE system for A-to-Y editing is highly desirable and will greatly expand the scope and potential for base editing in basic plant research and crop improvement. Nonetheless, such an BE has not been reported in plants to date. Here, we aimed to develop a series of plant AYBEs for A-to-Y base editing with improved performance. However, we only detected the occurrences of A-to-G, A-to-C, and A-to-T editing in rice protoplasts, and none of the stable lines showed A-to-C base transversions. Thus, we designated these BEs as plant A-to-K (K = G or T) base editors, or pAKBEs. Four pAKBEs were developed by fusion of pABE8e with an hMPG/mhMPG. Coupling with a transactivation module, VP64, improved the performance of pAKBEs in A-to-K base editing, and A-to-G and A-to-T editing efficiencies were as high as 17.24% and 82.22% in stable rice lines.