Abstract: Pre-messenger RNA (pre-mRNA) splicing is a critical mechanism for post-transcriptional regulation in plants. Through alternative splicing, plants produce diverse transcriptomes and proteomes that finely regulate development as well as responses to biotic and abiotic stresses. However, modulating the generation of specific splicing isoforms for functional characterization remains challenging, particularly in the non-model crop potato. Here, we show that two optimized TadA-derived base editors efficiently induce diverse mRNA splice variants by targeting specific splice sites. By evaluating multiple adenosine deaminases and performing multi-dimensional optimization, we developed an efficient adenine base editor RTF-ABE8e for potato. RTF-ABE8e achieved 100% editing efficiency at two StDL1 target sites in stable transgenic potato, with homozygous editing frequencies as high as 93.3% and 91.1%, respectively. We also developed RTF-TadDE, a dual-base editor based on a TadA-derived dual deaminase, for A-to-G and C-to-K (K = T/G) mutations in potato with an overall editing efficiency comparable to that of RTF-ABE8e. By targeting different splice sites with these base editors, we obtained diverse splicing isoforms carrying premature termination codons (PTCs) at StDL1 and StPDS and robust mutant phenotypes. These base editors enable efficient and precise editing of splice sites to trigger missplicing, making them powerful tools for manipulating splicing in plants.

Key words: adenine base editor, dual-base editor, splice sites, splicing manipulation