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.

中文翻译：

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甘蓝变种中GIGANTEA的无DNA诱变。使用CRISPR / Cas9核糖核蛋白复合物的人参

CRISPR–Cas9系统是编辑特定植物基因组中感兴趣基因的强大工具。确实，基因组编辑系统已用于增强多种农业特性并研究许多植物物种的基因功能，并且据报道，质粒介导的Cas9和单向导RNA（sgRNA）向植物的传递可促进高效编辑。然而，质粒DNA序列随机且稳定地整合到植物基因组中会引起插入诱变，并且需要额外的步骤来从编辑的植物基因组中去除这种外源序列。因此，本研究的目的是调查直接递送纯化CRISPR-Cas9核糖核蛋白（RNP）到原生质体从卷心菜有效性（甘蓝变种实蝇），重要的十字花科蔬菜。以开花时间调节基因GIGANTEA（GI）为目标，以延迟开花时间和延长营养生长为目标。我们调查了使用目标深度测序的目标诱变插入和删除率。在感染的原生质体中，使用其中一种sgRNA（sgRNA2）实现的突变频率为2％。从44％（46/103）的原生质体衍生的愈伤组织中再生出芽。因此，获得了三个独立且完全无转基因的突变体，包括一个均质双等位基因系，其中两个GI等位基因均已成功编辑，从而产生了完整的GI淘汰赛线。这些结果表明，无转基因的CRISPR-Cas9系统是改善甘蓝的农业有益性状的有前途的工具。