Knockout of transcription factor MYB28 by CRISPR/Cas9 for reducing glucosinolate content in rapeseed (Brassica napus L.)

Abstract: Rapeseed cake is a protein-rich by-product from the rapeseed oil extraction process. It contains high levels of glucosinolates (GSLs), which possess anti-nutritional functions after being hydrolyzed. Moreover, the hydrolyzed products of GSLs produce an extremely bitter taste. These adverse properties hinder the use of seedcake for human consumption and restrict its use in livestock feed manufacturing. The GSLs, especially aliphatic GSLs, significantly contribute to importing adverse properties compared to aromatic and indole GSLs. Thus, this study focused on partially down-regulating the aliphatic GSL biosynthesis by inducing targeted mutagenesis in the MYB28 transcription factor genes using CRISPR/Cas9. Six out of seven MYB28 paralogs were identified in the cv. Kumily genome of rapeseed and two sgRNAs were designed at the transcriptional regions for both MYB-type HTH domains of MYB28. The ribonucleoprotein (RNP), in which sgRNAs and Cas9 are mixed as a complex, was introduced into the rapeseed protoplasts. The transfected protoplasts were maintained for callus and shoot formation under in vitro conditions. For screening of mutation lines, genomic DNA was extracted from the true leaves of three regenerated shoots and seven randomly selected callus samples. The sgRNA target sites of five paralogs were amplified using gene-specific primers and screened for the presence of mutations. Screening results showed a single nucleotide insertion in one MYB28 paralog, LOC106428039, in one callus. This confirms that at least one selected sgRNA was successful inducing mutation in MYB28 using the RNA-based CRISPR-edited method.