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

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.