CRISPR/Cas9-mediated BoaAOP2s editing alters aliphatic glucosinolate side-chain metabolic flux and increases the glucoraphanin content in Chinese kale

• Three BoaAOP2s were isolated and edited using CRISPR/Cas9 system in Chinese kale. • The boaaop2 mutants of T1 generations were obtained in Chinese kale. • The content of glucoraphanin was increased and gluconapin was decreased in mutants. • Expression of upstream genes of glucosinolate biosynthesis were decreased in mutants. • BoaAOP2.1 is an effective gene for the alkenylation of aliphatic glucosinolates. Glucoraphanin (GRA) is an aliphatic glucosinolate (GSL), and its hydrolysis product has powerful anticancer activity. ALKENYL HYDROXALKYL PRODUCING 2 ( AOP2 ) gene, encodes a 2-oxoglutarate-dependent dioxygenase, which can catalyze GRA to form gluconapin (GNA). However, GRA only present in trace amounts in Chinese kale. To increase the content of GRA in Chinese kale, three copies of BoaAOP2 were isolated and edited using CRISPR/Cas9 system. The content of GRA was 11.71- to 41.29-fold (0.082–0.289 μmol g −1 FW) higher in T1 generation of boaaop2 mutants than in wild-type plants, and this was accompanied by an increase in the GRA/GNA ratio and reductions in the content of GNA and total aliphatic GSLs. BoaAOP2.1 is an effective gene for the alkenylation of aliphatic GSLs in Chinese kale. Overall, targeted editing of CRISPR/Cas9-mediated BoaAOP2s altered aliphatic GSL side-chain metabolic flux and enhanced the GRA content in Chinese kale, suggesting that metabolic engineering of BoaAOP2s has huge potential in improving nutritional quality of Chinese kale.