A Cas9-Based Toolkit to Program Expression Context in Saccharomyces Cerevisiae

From bread, beer and wine to synthetic biology, Saccharomyces cerevisiae has long been our go-to eukaryotic host. More recently, CRISPR/Cas9-directed homologous recombination has allowed for rapid and efficient chromosomal integration of heterologous genes and pathways. What remains relatively unexplored is how best to choose gene expression parts—integration locus, promoter, etc.—for a gene of interest. Here we develop a toolkit of well-characterized parts to program gene expression. Our toolkit consists of (1) two dozen genomic integration loci and (2) four dozen native promoters characterized at different growth phases and in different media; (3) modular protein tags to control protein expression level, solubility, and sub-cellular localization; and (4) a web tool to use these data to design donor DNA cassettes. These integration cassettes can be generated in 1-2 rounds of PCR and integrated with high efficiency using pre-cloned Cas9-gRNA plasmids targeting our integration sites. We demonstrate using our toolkit to program various expression contexts for genes of interest as a way to develop and optimize engineered strains.