New base editors facilitate C-to-G transversions in Nicotiana benthamiana

Base editors, including cytosine base editor (CBE) and adenine base editor, are widely used in plants to induce base transitions. Recently, glycosylase base editor (GBE) have been developed to induce C-to-G transversions but within a narrow editing window in human cells. Here, we describe six plant GBEs (GBE1-GBE6) composed of Cas9 nickase, human APOBEC3A, Escherichia coli-derived uracil DNA N-glycosylase (eUNG), and Arabidopsis thaliana-derived UNG (AtUNG). Our results illustrate that removing the uracil glycosylase inhibitor from CBE significantly enhances the C-to-G editing efficiency, making it on average 10.4 times higher than that of CBE, while concurrently reducing the C-to-T editing frequency. Additionally, both eUNG and AtUNG further suppress the C-to-T conversions induced by GBE2, GBE3, and GBE4. Notably, AtUNG enhances the purity of C-to-G transversions induced by GBE4 compared to GBE1. Furthermore, GBE1 and GBE4 exhibit a broader editing window (spanning positions 3 to 17). The expanded editing scope and improved efficiency of GBE4 make it a valuable tool for high-purity C-to-G transversions in Nicotiana benthamiana. The wide editing window of GBE1 and GBE4 establishes them as promising platforms for precise molecular engineering and crop genetic improvement, providing valuable insights for optimizing GBE-based genome editing systems in plants.