Base editing enables precise substitution of single nucleotides and is a promising technology for improvement of agronomic traits, including those of potato (Solanum tuberosum), the third most important food crop globally. Here, we developed efficient cytosine base editors (CBEs) for potato via multidimensional optimization. By evaluating several highly active cytidine deaminases, we identified two deaminases with high efficiency and distinct characteristics, which were used to construct CBEs for divergent editing applications. Furthermore, the editing efficiency of CBEs was significantly enhanced through several strategies: employing the Arabidopsis thaliana RPS5A promoter and the tobacco mosaic virus Ω enhancer to boost the expression of the editing reagents, fusing chromatin-modulating peptides, and co-expressing the human RNA m6A demethylase gene hFTO to enhance chromatin accessibility. In a hairy root assay, an efficient optimized base editor, RF-Sdd7-HNHN, elevated the average editing efficiency nearly 3-fold, from 26.8% (pre-optimization) to 76.7%. Using RF-Sdd7-HNHN, we achieved an 85.7% efficiency for simultaneous editing of target nucleotides in ACETOLACTATE SYNTHASE 1 (StALS1) and StALS2 and obtained homozygous mutant potato germplasm exhibiting herbicide resistance. In conclusion, this research established new and efficient CBEs for potato and offers insights and strategies for optimizing other CRISPR-based editing tools.

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