Improving plant C-to-G base editors with a cold-adapted glycosylase and TadA-8e variants

• Published in: Trends in biotechnology (Regular ed.) Vol. 43; no. 7; pp. 1765 - 1787
• Main Authors: Jiang, Yingli, Xiao, Zhi, Luo, Zhaopeng, Zhou, Suhuai, Tong, Chaoyun, Jin, Shan, Liu, Xiaoshuang, Qin, Ruiying, Xu, Rongfang, Pan, Lang, Li, Juan, Wei, Pengcheng
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.07.2025; Elsevier Limited
• Subjects: Adenine; Animals; base editing; Cold Temperature; CRISPR; CRISPR-Cas Systems; Cytidine Deaminase - genetics; Cytidine Deaminase - metabolism; Cytosine; Cytosine - metabolism; DNA glycosylase; Editing; Efficiency; Enzymes; Gene Editing - methods; Gene sequencing; Genomic analysis; Glycosylation; Guanine - metabolism; Humans; Internal Medicine; Mutation; Oryza - genetics; Plant breeding; plants; Plants, Genetically Modified - genetics; Proteins; Soybeans; TadA variants; Tobacco; UNG; Uracil; Uracil-DNA glycosidase; Uracil-DNA Glycosidase - genetics; Uracil-DNA Glycosidase - metabolism; Whole genome sequencing; TadA variants; CRISPR; base editing; plants; UNG
• ISSN: 0167-7799; 1879-3096; 1879-3096
• DOI: 10.1016/j.tibtech.2025.03.001

A cod uracil DNA glycosylase from the cold-adapted organism Gadus morhua enhanced cytosine (C)-to-guanine (G) base editor (CGBE) activities in rice.TadA-8e-derived cytidine deaminase (TadA-CDc) variants enabled rice C-to-G conversions.CDc-CGBEco achieved highly efficient C-to-G editing in rice, soybean, and tobacco.No significant off-target effects of the base editors derived from TadA-CDc were detected in rice. Plant cytosine (C)-to-guanine (G) base editors (CGBEs) have been established but suffer from limited editing efficiencies and low outcome purities. This study engineered a cod uracil DNA glycosylase (cod UNG, coUNG) from the cold-adapted fish Gadus morhua for plant CGBE, demonstrating 1.71- to 2.54-fold increases in C-to-G editing efficiency compared with the CGBE using human UNG (hUNG). Further engineering took advantage of TadA-8e-derived cytidine deaminases (TadA-CDs). These variants induced C substitutions with efficiencies ranging from 26.28% to 30.82% in rice cells, whereas adenine (A) conversion was negligible. By integrating coUNG and TadA-CDc elements with SpCas9 nickase, the resulting CDc-CGBEco achieved pure C-to-G editing without byproducts in up to 52.08% of transgenic lines. Whole-genome sequencing (WGS) analysis revealed no significant off-target effects of the CDc-BEs in rice. In addition, CDc-CGBEco enabled precise C-to-G editing in soybean and tobacco. These engineered CGBEs enhanced editing efficiency, purity, and specificity, suggesting their broad potential for applications in scientific research and crop breeding. [Display omitted] CDc-CGBEco introduced in this study is currently at a Technology Readiness Level (TRL) of 5. Using CDc-CGBEco, we obtained C-to-G-edited T0 rice plants with improved agronomic traits, such as herbicide resistance; we also found germline transmission of pure C-to-G edits in T1 offspring in the field. However, several challenges remain. For instance, limited editing activity in tobacco suggests that the universal applicability of CDc-CGBEco needs to be enhanced. In addition, although whole-genome sequencing was conducted to assess off-target effects, more long-term monitoring and diverse condition evaluations are necessary to comprehensively verify the safety and stability of this technology. A series of uracil DNA glycosylases (UNG) and cytidine deaminases were screened for upgrading plant cytosine (C)-to-guanine (G) base editors (CGBE). An engineered CDc-CGBEco fusing with a cod UNG (coUNG) and a TadA-8e-derived cytidine deaminases (TadA-CDc) exhibited superior performance in C-to-G editing, showing reliable and heritable base editing in monocots and dicots.