Browsing by Subject "CRISPR"

Saccharomyces cerevisiae is a popular organism in the production of biofuels, chemicals and pharmaceuticals. This is thanks to a good understanding of its metabolism, GRAS status and the ease of modification. Traditionally its genetic modification has been based on the use of selectable markers. Modifying multi gene pathways has required a sequential process consisting of multiple single gene disruptions together with marker recycling. Additionally, many industrial S. cerevisiae strains are polyploid and lack the same tools for their modification as laboratory strains. In this study we sought to develop CRISPR/Cas9 based genetic engineering method for the modification of industrial S. cerevisiae strains. The CRISPR/Cas9 system is based on the adaptive immunity system of bacteria. It makes use of the Cas9 endonuclease which produces double stranded DNA brake to any location determined by a gRNA molecule. This causes the activation of DNA repair mechanisms which can be utilized to for the genomic integration of a template DNA. This makes transformation events much more likely and thus enables producing multiple modifications at once and removes the need for the of use selectable markers. In our approach Cas9 and gRNA were transformed into the cell in a plasmid together with a separate template DNA molecule. We used this method to remove lyp1, ura3 and can1 genes from diploid and polyploid industrial S. cerevisiae strains multiple genes at a time. Simultaneously we evaluated the effect of the NHEJ repair mechanism on CRISPR/Cas9 by repeating the tests with a deletion strain missing the ku70 gene required by NHEJ. Finally the method was used for the metabolic engineering by integrating the five gene violacein metabolic pathway into two loci in a single transformation event. This study demonstrated the CRISPR/Cas9 method is well suited for the modification of industrial S. cerevisiae strains and is capable of modifying up to three loci at a time in a polyploid yeast strain.