CANCELED: DNA-free Multiplex CRISPR/Cas9 Editing of BnGTR Reduces Seed Glucosinolate Content in Rapeseed (Brassica napus L.)

The accumulation of glucosinolates (GSLs) in rapeseed (Brassica napus L.) seeds limits their use as a protein-rich meal for human and animal consumption due to anti-nutritional effects. While conventional breeding has reduced seed GSL content (SGC), further reductions are necessary for broader utilization. In this study, we employed DNA-free CRISPR/Cas9-mediated multiplex gene editing, targeting glucosinolate transporter genes (BnGTR1 and BnGTR2) to reduce SGC in rapeseed. We designed specific sgRNAs targeting 12 BnGTR paralogs and delivered preassembled Cas9-sgRNA ribonucleoprotein (RNP) complexes into rapeseed protoplasts via polyethylene glycol (PEG)-mediated transfection. We evaluated mutagenesis efficiency, editing types, and SGC reductions using high-resolution fragment analysis, Sanger/amplicon sequencing, and LC-MS quantification, respectively. RNP-mediated gene editing achieved a mutation efficiency of 80.95%, generating mutants with significant reductions in total SGC (56%) compared to wild-type plants. The aliphatic glucosinolates, particularly progoitrin and epi-progoitrin, decreased by 57%, while indolic glucosinolates increased by 16-fold, indicating a shift in glucosinolate composition due to gene editing. Frameshift mutations and large deletions were predominant, suggesting effective disruption of GSL transporter activity. A comparative analysis with plasmid-mediated CRISPR/Cas9 editing highlighted superior efficiency, specificity, and transgene-free editing achieved through RNP delivery. Our findings demonstrate the potential of multiplex CRISPR/Cas9 RNP-mediated gene editing as a precise, scalable, and regulatory-friendly approach for reducing SGC in polyploid crops like rapeseed, paving the way for the development of nutritionally enhanced oilseed varieties.