DNA-free mutagenesis of GIGANTEA in Brassica oleracea var. capitata using CRISPR/Cas9 ribonucleoprotein complexes

The CRISPR-Cas9 system is a powerful tool for editing genes of interest in specific plant genomes. Indeed, genome-editing systems have been used to enhance a variety of agricultural traits and to study gene functions in many plant species, and the plasmid-mediated delivery of Cas9 and single guide RNA (sgRNA) to plants has been reported to facilitate highly efficient editing. However, the random and stable integration of plasmid DNA sequences into plant genomes can cause insertional mutagenesis, and an additional step is required to remove such foreign sequences from edited plant genomes. Accordingly, the aim of the present study was to investigate the effectiveness of directly delivering purified CRISPR-Cas9 ribonucleoproteins (RNPs) to protoplasts from cabbage (Brassica oleracea var. capitata), an important cruciferous vegetable. The flowering-time regulator gene GIGANTEA (GI) was targeted, with the goal of delaying flowering time and prolonging vegetative growth. We investigated the targeted mutagenesis insertion and deletion rates using targeted deep sequencing. The mutation frequency achieved using one of the sgRNAs (sgRNA2) was 2% in the infected protoplast. The shoots were regenerated from 44% (46/103) of protoplast-derived calli. Consequently, three independent and completely transgene-free mutants were obtained, including one homogeneous biallelic line in which both GI alleles were successfully edited, thereby yielding a complete GI knockout line. These results suggest that the transgene-free CRISPR-Cas9 system is a promising tool for improving agricultural beneficial traits of cabbage.