A Crispr/Cas9 Mediated Genome Engineering – Examples for Heterologous Carotenoid Production in Saccharomyces Cerevisiae

One of the key drivers for successful metabolic engineering is the efficiency by which genomes can be edited. CRISPR/Cas9 systems have quickly become an important new method for precision genome engineering of yeast and many other species. For example, the introduction of designed double-stranded breaks in S. cerevisiae allows for rather efficient â??marker-freeâ?? recovery of cells which have been modified by donor-DNA mediated homologous recombination. Although, various strategies have been published for the use of CRISPR/Cas9 systems in yeast, there is room for further improvement. Key elements of a CRISPR/Cas9 system are the expression of Cas9, the design and expression of the sgRNA(s), and the design and introduction of donor DNA fragments. We present various novel strategies for the introduction and use of Cas9, sgRNA and donor DNA to efficiently introduce mutations and heterologous pathways as well as the deletion of genomic DNA fragments. The introduction of a carotenoid pathway with three genes from Xanthophyllomyces dendrorhous in Saccharomyces cerevisiae is our testbed. Both singleplex and multiplex approaches will be demonstrated. The resulting carotenoid producing strains are characterized using proteomics and direct product measurements.