Knockout of fatty acid elongase1 homeoalleles in amphidiploid Brassica juncea leads to undetectable erucic acid in seed oil

Indian mustard (Brassica juncea L.) is a major oilseed crop with significant economic and nutritional importance within the Indian subcontinent. While its seed oil offers valuable dietary benefits, including a balanced ratio of human essential fatty acids, the traditional high oil-yielding varieties contain an elevated level of erucic acid (EA, C22:1) that is associated with adverse health effects. Therefore, developing low erucic acid (LEA) mustard cultivars is crucial for broader utilization and consumer safety. In this study, we employed CRISPR/Cas9 genome editing tools to disrupt the fatty acid elongase1 (FAE1) gene that encodes a key enzyme in EA biosynthesis in two high erucic acid (HEA) B. juncea cultivars, PCR7 and JD6. Targeted knockout (KO) of BjFAE1 homeoalleles (BjFAE1.1 and BjFAE1.2) in this amphidiploid plant using CRISPR/Cas9 constructs, each carrying two guide RNAs led to monoallelic and biallelic mutations. Biallelic KO lines showed a near-complete elimination of EA (<0.5% in PCR7, undetectable in JD6) with a significant increase in nutritionally beneficial oleic acid ([~]30% in PCR7, [~]35% in JD6), while the content of essential fatty acids also increased significantly, suggesting rerouting of carbon flux from EA biosynthesis. Importantly, these LEA lines retained key agronomic traits like plant seed yield and oil content, matching the productivity of the control elite cultivars. Our findings underscore the effectiveness of CRISPR/Cas9 technology for editing B. juncea genome, producing LEA seed oil lines with improved nutritional quality and thus expanding the applications of this important oilseed crop.