High-Efficiency and Multiplex Adenine Base Editing in Plants Using New TadA Variants

Recently reported adenine base editors (ABEs) exhibit powerful potentials in targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function study and crop breeding. However, editing efficiency varies significantly among different target sites. Here we investigated the activities of three evolved E. coli adenosine deaminase TadA variants (TadA8e, TadA8.17, and TadA8.20) side-by-side in transgenic rice. We found that TadA8e outperforms TadA8.17 and TadA8.20, and induces efficient A-to-G conversion at all tested sites in the rice genome, including those uneditable for ABE7.10 in our previous experiments. Furthermore, V82S/Q154R mutations were incorporated into TadA8e, resulting in a new variant that we named TadA9. Our data shows that TadA9 is broadly compatible with CRISPR/SpCas9, CRISPR/SpCas9-NG, CRISPR/SpRY as well as CRISPR/ScCas9 nickase systems, achieving comparable or enhanced editing in a larger editing window at diverse PAM sites as compared to TadA8e. Finally, TadA9 was employed to simultaneously install novel SNPs in four endogenous herbicide-target genes in the commercial rice cultivar Nangeng 46 for the potential field application in weed control. In conclusion, we successfully generate a series of novel ABEs which can efficiently edit adenosines in the rice genome to a great extent, and our finding implies the promising application of TadA9 as well as TadA8e in other plant base editor design and crop molecular breeding.