Fusing T7 exonuclease with the G-to-T base editor enhances the editing purity in rice.

The G-to-T base editor (GTBE) is a genome editing tool that does not rely on deaminases. However, its application has been limited by low G-to-T editing efficiency and high insertion and deletion (InDel) byproduct rates. In this study, we develop a series of editors (GTBE1-GTBE7) using three strategies: modification of the Cas9 nickase (nCas9) variant, fusion with a transcriptional activation domain, and fusion with a 5'-3' exonuclease targeting nicked DNA. Both protoplast and stable rice transformant analyses reveal that the GTBE7 editor, which incorporates T7 exonuclease, significantly improves G-to-T editing purity compared with the previously reported GTBE1 editor. In stable transformants, the InDel efficiency of GTBE7 is reduced by 14%-27.7% compared with GTBE1. To further broaden the editing range of GTBE7, we develop the GTBE7_nSpRY editor, enabling G-to-T base editing at non-NGG protospacer adjacent motif (PAM) sequences. Using GTBE7_nSpRY, we create herbicide-resistant rice and identify the G628A mutation site, generating imazethapyr-resistant rice plants through G-to-C byproduct edits. In summary, the GTBE7 and GTBE7_nSpRY editors developed in this study enhance G-to-T editing purity, reduce InDel byproduct efficiency, and offer potential for expanding the application of G-to-T base editing in plants.