A method called Cas-3P allowing for immediate, multiplexed and sequential genome engineering was developed using one plasmid expressing Cas9 and three marked plasmid backbones (P1, P2 and P3) for guide RNA (gRNA) expression. The three marked gRNA plasmid backbones were recurred in a P1-P2-P3 order for sequential gene targeting, without construction of any additional plasmid and elimination of gRNA plasmid by induction in each round. The efficiency of direct gRNA plasmid curing mediated by Cas-3P was more than 40% in sequential gene targeting. Besides, Cas-3P allowed single-, double- and triple-loci gene targeting with an efficiency of 75%, 36.8% and 8.2% within 3-4 days, respectively. Through three sequential rounds of gene targeting within 10 days, S. cerevisiae was optimized for the production of patchoulol by replacing one promoter, overexpressing three genes and disrupting four genes. The work is important for practical application in the cell factory engineering of S. cerevisiae.
Keywords: CRISPR/Cas9; Cell factory; Gene targeting; Saccharomyces cerevisiae.