Evaluation of Bna.SCT and Bna.REF1 as Target Genes to Reduce Sinapine in Rapeseed Using a Protoplast-Based CRISPR RNP...

Rapeseed is a major oil crop worldwide, producing both oil and a high amount of protein. However, the use of its seed meal as a protein source for animal feed is limited by antinutritional factors, such as sinapine, which reduces nutrient absorption and affects the palatability. Efforts to reduce sinapine levels through conventional breeding have had limited success. Given the challenges of a changing climate and a growing global population, maximising crop utility, particularly the value of seed meal as a byproduct, is increasingly important. Genetic modification has been successfully used to reduce sinapine in rapeseed, but regulatory restrictions limit its commercial adoption in some regions. CRISPR-Cas gene editing, which is gaining broader global acceptance, offers a promising alternative to directly produce transgene-free mutants. In this study, we build on our previous work by generating transgene-free rapeseed mutants using protoplast-based CRISPR RNP gene editing. We successfully targeted the Bna.SCT and Bna.REF1 genes with editing efficiencies of 22%-63%, frequently achieving mutations in all four alleles of the target genes in T₂ plants with a single sgRNA. Seed sinapine content was reduced by up to 38% in Bna.SCT mutants and 77% in Bna.REF1 mutants, with no observed effects on plant growth or development. These findings suggest that Bna.REF1 is the most effective target for sinapine reduction in transgene-free mutants among the genes tested in our lab.