Figure 7

CRISPR-Cas system constructed based on autonomously replicating plasmids with MAM1 sequence. Leveraging the instability of autonomously replicating plasmid with an AMA1 sequence, the plasmid will be lost through subculture without resistance and the strain with lost plasmid no longer has the resistance gene of plasmid. This allows the resistance gene to be reused in subsequent transformations. Red circles indicate the transformant containing hyg obtained by ATMT; blue circles indicate wild-type strains (control).