RNA Polymerase III Promoters Compatible with CRISPR Gene Regulation in <i>Saccharomyces cerevisiae</i>

<i>Saccharomyces cerevisiae</i>is a model organism commonly used to study gene regulation and function recently via CRISPR-(<i>d</i>)Cas9 technologies. Modulating the expression of multiple gene targets simultaneously is often important for synthetic biology and metabolic engineering applications and is crucial for genetic interaction studies. CRISPR-based systems can be used to target multiple genetic loci via expression of multiple single-guide RNAs (sgRNAs) in a single cell. However, there are currently a limited number of well-characterized RNA polymerase III (Pol III) promoters (e.g., pSNR52) for sgRNA expression in <i>S. cerevisiae</i>. Herein, we characterize 20 RNA Pol III promoters from different yeast species, from <i>S. cerevisiae</i> itself or from mammals, for their utility toward effectively mediating CRISPR activation and repression in <i>S. cerevisiae</i>. We show that the Pol III promoter cross-species functionality is impacted by promoter architecture and inclusion of core sequence motifs and that scaffold-mediated recruitment of multiple effectors can rescue poor promoter function in some contexts. Also, we highlight two <i>Kluyveromyces lactis</i> Pol III promoters that mediate CRISPR function as well as the gold standard <i>S. cerevisiae</i> pSNR52 and previously described tRNA promoters. Finally, we show that these non-native promoters enable effective simultaneous CRISPR-mediated activation and repression of endogenous <i>S. cerevisiae</i> genes to enhance resistance to hydrogen peroxide. The Pol III promoters described here highlight the cross-species compatibility of genetic units in simple eukaryotes and will be useful for synthetic biology and phenotype engineering applications in yeast.