Fig. 2

The schematic of gene editing using CRISPR/Cas9 system mediated by recyclable plasmid P426-CL. (i) single gRNA expression plasmid was constructed based on P426-CL. The recyclable gRNA expression plasmid was transformed into cells containing Cas9 protein to perform gene editing with homologous donor DNA. (ii) after successful gene editing, genetically modified strain was cultivated in galactose medium to induce expression of Cre recombinase. The 2μ replication origin between two loxP loci in gRNA expression plasmid was excised by Cre recombinase. The gRNA expression plasmid was lost in cells along with passage. (iii) the yeast culture in galactose medium was plated onto SD-glucose/5-FOA agar plate. Cells without gRNA expression plasmid grew out and were selected for preparation of competent cell. The new gRNA expression plasmid could be transformed into the competent cell for next round of gene editing