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 considerable economic and nutritional importance globally. While its seed oil offers valuable dietary benefits due to a balanced ratio of human essential fatty acids, the traditional high oil-yielding varieties contain an elevated level of erucic acid (EA, C22:1) associated with adverse health effects. Therefore, developing low erucic acid (LEA) mustard cultivars is crucial for broader utilization and consumer safety. In this study, CRISPR/Cas9 genome editing tool was employed 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 (∼39% EA) and JD6 (∼45% EA). Targeted knockout (KO) of BjFAE1 homeoalleles (BjFAE1.1 and BjFAE1.2) in this amphidiploid plant species using CRISPR/Cas9 constructs, each carrying two guide RNAs led to generation of single (either fae1.1 or fae1.2) and double (fae1.1fae1.2) mutants. Best performing homozygous fae1.1fae1.2 KO lines showed a near-complete elimination of EA in both the cultivars (<0.5% in PCR7, undetectable in JD6) with a marked increase in nutritionally beneficial oleic acid (from ∼18% to ∼32% in PCR7, from ∼9% to ∼38% in JD6). Moreover, the content of essential fatty acids also increased substantially [linoleic acid (C18:2) 1.9-fold in PCR7 and 2.1-fold in JD6; linolenic acid (C18:3) 2.5-fold in PCR7 and 1.4-fold in JD6], 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 unedited control elite cultivars. Our findings underscore the effectiveness of CRISPR/Cas9 technology for editing B. juncea genome, developing plant lines producing LEA seed oil with improved nutritional quality and broadening the utility of this important oilseed crop for food and non-food applications.