Efficient genome editing using CRISPR-Cas9 RNP delivery into cabbage protoplasts via electro-transfection

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.