MAD7-mediated genome editing via Agrobacterium enables recovery of hexa-allelic edited lines carrying distinct mutations in all six gene copies in wheat (Triticum aestivum L.) T0 plants and stable inheritance of edits into the following generation

Abstract The expansion of genome-editing tools for bread wheat ( Triticum aestivum L.) is essential to accelerate trait improvement while addressing intellectual property constraints associated with widely used CRISPR systems. MAD7 (ErCas12a), a royalty-accessible CRISPR nuclease, represents a potential alternative to CRISPR-Cas9; however, its performance in complex polyploid crops remains insufficiently characterized. In this study, the in planta genome-editing efficiency of MAD7 in hexaploid wheat was evaluated using Agrobacterium -mediated transformation. Conserved coding regions of the TaLCYε ( LYCOPENE EPSILON CYCLASE ) gene, a key regulator of carotenoid flux, were targeted across all three wheat subgenomes (A, B, and D). MAD7-mediated editing showed strong dependence on protospacer adjacent motif (PAM) composition, with detectable activity only at a T-rich PAM (TTTG), resulting in targeted mutations in 26% of transgenic T 0 plants. Notably, MAD7 enabled recovery of hexa-allelic edited lines carrying distinct mutations in all six gene copies already in the T 0 generation, with edits stably inherited in the T 1 progeny and evidence of continued nuclease activity across generations. In contrast, CRISPR-Cas9 achieved higher editing efficiencies under experimental conditions for two of three guide RNAs tested (up to 86%) and produced a broader spectrum of mutations, predominantly small insertions and deletions. MAD7-induced edits were characterized mainly by medium-sized deletions (6–15 bp), consistent with Cas12a-type staggered cleavage. This is the first study that demonstrated recovery of hexa-allelic MAD7-edited mutants in hexaploid wheat T 0 plants via Agrobacterium -mediated transformation, with stable inheritance into T 1 .