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The clustered regulatory interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) system has developed into a powerful gene-editing tool that has been successfully applied to various plant species. However, studies on the application of the CRISPR/Cas9 system to cultivated  Brassica  vegetables are limited. Here, we reported CRISPR/Cas9-mediated genome editing in Chinese kale ( Brassica oleracea  var.  alboglabra ) for the first time. A stretch of homologous genes, namely  BaPDS1  and  BaPDS2 , was selected as the target site. Several stable transgenic lines with different types of mutations were generated via  Agrobacterium -mediated transformation, including  BaPDS1  and  BaPDS2  double mutations and  BaPDS1  or  BaPDS2  single mutations. The overall mutation rate reached 76.47%, and these mutations involved nucleotide changes of fewer than 10 bp. The clear albino phenotype was observed in all of the mutants, including one that harbored a mutation within an intron region, thereby indicating the importance of the intron. Cleavage in Chinese kale using CRISPR/Cas9 was biased towards AT-rich sequences. Furthermore, no off-target events were observed. Functional differences between  BaPDS1  and  BaPDS2  were also assessed in terms of the phenotypes of the respective mutants. In combination, these findings showed that CRISPR/Cas9-mediated targeted mutagenesis can simultaneously and efficiently modify homologous gene copies of Chinese kale and provide a convenient approach for studying gene function and improving the yield and quality of cultivated  Brassica  vegetables.

CRISPR/Cas9-mediated mutagenesis of homologous genes in Chinese kale