CRISPR–Cas system enables fast and simple genome editing of industrial Saccharomyces cerevisiae strains

• Published in: Metabolic engineering communications 2015-12, Vol.2, p.13-22
• Main Authors: Stovicek, Vratislav, Borodina, Irina, Forster, Jochen
• Format: Article
• Language: English
• ISSN: 2214-0301; 2214-0301
• DOI: 10.1016/j.meteno.2015.03.001

There is a demand to develop 3rd generation biorefineries that integrate energy production with the production of higher value chemicals from renewable feedstocks. Here, robust and stress-tolerant industrial strains of Saccharomyces cerevisiae will be suitable production organisms. However, their genetic manipulation is challenging, as they are usually diploid or polyploid. Therefore, there is a need to develop more efficient genetic engineering tools. We applied a CRISPR–Cas9 system for genome editing of different industrial strains, and show simultaneous disruption of two alleles of a gene in several unrelated strains with the efficiency ranging between 65% and 78%. We also achieved simultaneous disruption and knock-in of a reporter gene, and demonstrate the applicability of the method by designing lactic acid-producing strains in a single transformation event, where insertion of a heterologous gene and disruption of two endogenous genes occurred simultaneously. Our study provides a foundation for efficient engineering of industrial yeast cell factories. • We developed CRISPR–Cas9-based system for gene disruptions in industrial yeast. • We showed high rate of disruption efficiency in unrelated industrial strains. • Gene knock-in may be performed simultaneously with gene disruption. • Use of the described Cas9-based system results in marker-free stable genetic modifications. • The method was applied for single-step construction of lactic acid-producing strains.