Plant Biotechnology Journal, 2025, https://doi.org/10.1111/pbi.70063 

Abstract

Nucleotide variations are essential genetic variants and play a key role in shaping crop genetic diversity and contributing to the development of superior agronomic traits. DNA deaminase-mediated cytosine and adenine base editors generate highly efficient C-to-T and A-to-G substitutions and have been widely applied in many plant species to correct defective alleles and create novel alleles by artificial evolution for rapid crop genetic improvement. Glycosylase-based guanine base editors were also developed in rice and converted the G to T. However, no direct thymine base editing for direct T editing in plants has been reported so far. Additionally, it remains necessary to explore DNA glycosylases and develop novel plant glycosylase-based base editors. In this study, we comprehensively investigated the base editing activities of several artificially evolved thymine DNA glycosylases (TDG-EKΔ, TDG3Δ, and gTBEv3) and cytosine DNA glycosylases (CDG4Δ and gCBEv2) in rice by fusing the glycosylases to the N-terminus of SpCas9n. All thymine DNA glycosylases successfully achieved T-to-G editing, and gTBEv3 demonstrated the highest T-to-G editing efficiency. CDG4Δ and gCBEv2 also achieved efficient and comparable direct C editing activity and the C-to-G and C-to-T editing events were the dominant outcomes. Besides the base editing, various nucleotide insertions and deletions were also generated by these evolved DNA glycosylases. Collectively, our results demonstrate that glycosylase-based thymine and cytosine base editors can produce diverse editing events in rice, providing a potential biological mutation platform for direct evolution of crop endogenous genes. 

Plant Biotechnology Journal，IF=10.1

https://onlinelibrary.wiley.com/doi/10.1111/pbi.70063