CRISPR-Cas9 disruption of flavanone 3-hydroxylase produces a green phenotype and alters flavone metabolites in allotetraploid perilla

Perilla frutescens var. crispa is a high-value horticultural crop known for its diverse bioactive metabolites, yet the molecular basis underlying its metabolic variation remains poorly understood for targeted metabolic engineering. In this study, we employed CRISPR–Cas9 to disrupt the flavanone 3-hydroxylase gene ( F3H ), a key branch-point enzyme in the flavonoid pathway. We generated stable, T-DNA-free null-segregant lines that exhibited a visible transition from red to green leaves. Metabolite profiling across multiple independently derived edited lines showed that F3H disruption markedly reduced anthocyanin accumulation and was consistently associated with increased levels of flavone-related metabolites, including an approximately six-fold increase in luteolin content compared to the wild type. Transcriptome analysis revealed changes in the expression patterns of phenylpropanoid and flavonoid biosynthetic genes consistent with the observed metabolic profiles. In addition, rosmarinic acid content was elevated in the edited lines, suggesting that F3H disruption may affect broader phenylpropanoid metabolism. Together, our findings provide functional insights into F3H in perilla and highlight the potential of targeted gene editing for modifying metabolite composition in this polyploid crop.