Draft genome sequence of the Saccharomyces cerevisiae SpyCas9 expressing strain IMX2600, a laboratory and platform strain from the CEN.PK lineage for cell-factory research.

The biobased-economy aims to create a circular biotechnology ecosystem to transition from a fossil fuel-based to a sustainable industry based on biomass. For this, new microbial cell-factories are essential. We present the draft genome of the CEN.PK-derived Saccharomyces cerevisiae SpyCas9 expressing strain (IMX2600), that serve as chassis of new cell-factories.

Expanding the genome editing toolbox of Saccharomyces cerevisiae with the endonuclease ErCas12a.

ErCas12a is a class 2 type V CRISPR-Cas nuclease isolated from Eubacterium rectale with attractive fundamental characteristics, such as RNA self-processing capability, and lacks reach-through royalties typical for Cas nucleases. This study aims to develop a ErCas12a-mediated genome editing tool applicable in the model yeast Saccharomyces cerevisiae. The optimal design parameters for ErCas12a editing in S. cerevisiae were defined as a 21-nt spacer flanked by 19 nt direct repeats expressed from either RNApolII or III promoters, achieving near 100% editing efficiencies in commonly targeted genomic locations. To be able to transfer the ErCas12a genome editing tool to different strain lineages, a transportable platform plasmid was constructed and evaluated for its genome editing efficiency. Using an identical crRNA expression design, the transportable ErCas12a genome editing tool showed lower efficiency when targeting the ADE2 gene. In contrast to genomic Ercas12a expression, episomal expression of Ercas12a decreases maximum specific growth rate on glucose, indicating ErCas12a toxicity at high expression levels. Moreover, ErCas12a processed a multispacer crRNA array using the RNA self-processing capability, which allowed for simultaneous editing of multiple chromosomal locations. ErCas12a is established as a valuable addition to the genetic toolbox for S. cerevisiae.

Improving CRISPR-Cas9 mediated genome integration in interspecific hybrid yeasts.

Saccharomyces pastorianus is not a classical taxon, it is an interspecific hybrid resulting from the cross of Saccharomyces cerevisiae and Saccharomyces eubayanus. Exhibiting heterosis for phenotypic traits such as wort α-oligosaccharide consumption and fermentation at low temperature, it has been domesticated to become the main workhorse of the brewing industry. Although CRISPR-Cas9 has been shown to be functional in S. pastorianus, repair of CRISPR-induced double strand breaks is unpredictable and preferentially uses the homoeologous chromosome as template, preventing targeted introduction of the desired repair construct. Here, we demonstrate that lager hybrids can be edited with near 100% efficiency at carefully selected landing sites on the chimeric SeScCHRIII. The landing sites were systematically selected and evaluated for (i) absence of loss of heterozygosity upon CRISPR-editing, (ii) efficiency of the gRNA, and (iii) absence of effect on strain physiology. Successful examples of highly efficient single and double gene integration illustrated that genome editing can be applied in interspecies hybrids, paving the way to a new impulse to lager yeast strain development.