RNA Polymerase III Promoters Compatible with CRISPR Gene Regulation in .

is a model organism commonly used to study gene regulation and function recently via CRISPR-()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 . Herein, we characterize 20 RNA Pol III promoters from different yeast species, from itself or from mammals, for their utility toward effectively mediating CRISPR activation and repression in . 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 Pol III promoters that mediate CRISPR function as well as the gold standard pSNR52 and previously described tRNA promoters. Finally, we show that these non-native promoters enable effective simultaneous CRISPR-mediated activation and repression of endogenous 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.