CRISPR/Cas9-mediated knockout of DFR alters pigmentation and shifts flavonoid accumulation in red leaf lettuce without detectable growth penalties

Red leaf lettuce ( Lactuca sativa L. cv. ‘Red Fire’) is a preferred crop in plant factories with artificial light (PFALs) due to its short cultivation cycle and high anthocyanin content, which increases both its nutritional value and visual appeal. However, anthocyanins strongly influence leaf coloration and antioxidant profiles, and their levels are highly responsive to the light environment. Therefore, targeted editing of flavonoid biosynthesis may provide a breeding strategy to diversify pigment composition and associated functional traits under PFAL conditions. In this study, we used CRISPR/Cas9 to knock out DFR (dihydroflavonol 4-reductase), a key enzyme in the anthocyanin pathway. Genome-edited lines were generated via a dual-guide RNA system, resulting in a successfully edited red leaf genotype. The DFR -knockout lines displayed a complete loss of red pigmentation and a visibly distinct green phenotype. Metabolite profiling revealed a significant decrease in anthocyanin levels, accompanied by an increase in total flavonoid levels in some lines. Growth traits, including shoot dry weight and leaf number, were not significantly affected, suggesting that DFR knockout does not compromise growth under PFAL conditions. These findings highlight DFR as a promising target for creating pigment-altered lettuce lines for controlled-environment cultivation, including PFAL systems.