Nowadays, genome editing in plants has become much easier thanks to the recently developed clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR–Cas9) nuclease system. However, to combine protoplast technology with the CRISPR–Cas9 system in plants, a stable and an efficient foreign DNA delivery system is essential for gene editing. In the present study, we developed an electro-transfection system for CRISPR–Cas9 ribonucleoprotein (RNP) delivery to cabbage protoplasts. Under 1000 V treatment, the frequency of initial cell division and total number of cell colonies formed were 47.7 ± 2.5% and 52 ± 7.5%, respectively. The total number of cell colonies formed following 1000 V treatment was 1.4 times higher than that following polyethylene glycol (PEG) treatment. However, the frequency of initial cell division and total number of cell colonies formed from protoplasts decreased with increasing voltage. Cy3–Cas9 protein delivery into the nucleus was confirmed through both electro-transfection and PEG-mediated transfection using confocal laser scanning microscopy. The frequency of insertions and deletions in the synthesized guide RNA of phytoene desaturase 1 was the highest at 3.4% following electro-transfection at 1000 V with a pulse width of 20 ms and only 1.8% following PEG-mediated transfection. These results indicate that electro-transfection is more efficient in RNP delivery to protoplast than PEG-mediated transfection in cabbage for PDS1 sgRNA delivery. Therefore, the electro-transfection system developed in the present study presents the possibility it could be used for DNA-free genome editing of other crops.

如今，由于最近开发的簇状规则间隔的短回文重复序列（CRISPR）相关蛋白9（CRISPR–Cas9）核酸酶系统，植物的基因组编辑变得更加容易。然而，要将原生质体技术与CRISPR–Cas9系统结合起来，稳定而有效的外源DNA传递系统对于基因编辑至关重要。在本研究中，我们开发了一种用于CRISPR–Cas9核糖核蛋白（RNP）递送至甘蓝原生质体的电转染系统。在1000 V处理下，初始细胞分裂的频率和形成的细胞集落总数分别为47.7±2.5％和52±7.5％。1000 V处理后形成的细胞集落总数比聚乙二醇（PEG）处理后高1.4倍。然而，初始细胞分裂的频率和原生质体形成的细胞集落总数随着电压的升高而降低。使用共聚焦激光扫描显微镜通过电转染和PEG介导的转染都证实了Cy3–Cas9蛋白已进入细胞核。合成的八氢番茄红素去饱和酶1的指导RNA中的插入和删除的频率是最高的，在1000 V以20 ms的脉冲宽度电转染后为3.4％，而在PEG介导的转染后仅为1.8％。这些结果表明电转染比甘蓝中的PEG介导的转染对RNP传递到原生质体更有效。使用共聚焦激光扫描显微镜通过电转染和PEG介导的转染都证实了Cy3–Cas9蛋白已进入细胞核。合成的八氢番茄红素去饱和酶1的指导RNA中的插入和删除的频率是最高的，在1000 V以20 ms的脉冲宽度电转染后为3.4％，而在PEG介导的转染后仅为1.8％。这些结果表明电转染比甘蓝中的PEG介导的转染对RNP传递到原生质体更有效。使用共聚焦激光扫描显微镜通过电转染和PEG介导的转染都证实了Cy3–Cas9蛋白已进入细胞核。合成的八氢番茄红素去饱和酶1的指导RNA中的插入和删除的频率是最高的，在1000 V以20 ms的脉冲宽度电转染后为3.4％，而在PEG介导的转染后仅为1.8％。这些结果表明电转染比甘蓝中的PEG介导的转染对RNP传递到原生质体更有效。PDS1 sgRNA递送。因此，本研究开发的电转染系统提出了将其用于其他农作物的无DNA基因组编辑的可能性。