CRISPR/Cas12a-mediated allele engineering of SmAPRR2 and SmGLK2 reveals complementary control of fruit peel and flesh chlorophyll pigmentation in eggplant
A multiplex CRISPR/Cas12a system is established in eggplant accession MEL3, representing, to the authors' knowledge, the first application of this nuclease for genome editing in eggplant and demonstrating the potential of Cas12a for functional genomics, allele engineering, and precision breeding in eggplant.
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
.
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This study uncovers a rare case in which a deletion of just two amino acids is sufficient to generate a potent dominant-negative regulator, designated SmMYB1alf-D, which enables reliable prediction of fruit color and provides a breeding strategy to precisely manipulate anthocyanin metabolism.
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