Transgene-free gene editing of BnaCCD4 homologs significantly improves the carotenoid level of seeds in Brassica napus

• Rapeseed carotenoid content is typically low due to CCD4-mediated degradation. This study employs a novel CRISPR/Cas9 approach to inactivate multiple CCD4 gene copies, significantly boosting carotenoid levels without impacting seed quality or yield. • We achieved a more than 20-fold increase in β-carotenoid accumulation and revealed delayed carotenoid degradation during seed maturation and leaf senescence. • This research advances molecular techniques in crop biofortification, showing the potential for CRISPR/Cas9 to enhance nutritional value in oilseed crops. The findings also offer practical applications for improving the oxidative stability of rapeseed oil. The absorption of carotenoids, plant pigments with significant health benefits, is promoted when ingested with lipids. However, rapeseed carotenoid contents are relatively low. Our previous study identified that the inactivation of BnaC3.CCD4, a carotenoid cleavage dioxygenase which degrades carotenoids, in rapeseed petals, increased carotenoid accumulation. The present study created loss-of-function single, double, and triple mutants of three other CCD4 copies using a CRISPR/Cas9 system. The results showed that some double and triple mutants exhibited a more than 20-fold increase in seed β-carotene contents without harming quality or yield. In addition, the carotenoid content of BnaC1.ccd4 was close to those of some triple and double mutants. During seed maturation and leaf senescence, BnaCCD4 mutations delayed carotenoid degradation. The reduced power activities and hydroxyl radical scavenging activities of mutant seed oils indicated increased antioxidation. Enzyme activity analysis of BnaCCD4s in Arabidopsis ccd4 mutant seeds confirmed diverging oxidative cleavage activities on carotenoids.