plant biotechnology journal, 2025,
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
