Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a

A variety of methods have been developed for the repression of gene expression using CRISPR-Cas systems, where a catalytically dead Cas protein (dCas) is used to regulate genes in an RNA-guided manner. While dCas9 has been extensively studied, dCas12a, an RNA-programmable endonuclease is potentially better suited for gene regulation due to its preference for T-rich PAMs that allows it to more easily target AT-rich promoter sequences, and built-in RNase activity which can process a single crRNA array encoding multiple spacers into individual gRNAs, thereby simplifying multiplexed gene regulation. Here, we develop a flexible dCas12a-based CRISPRi system for S. cerevisiae and systematically evaluate its design features. This includes the role of NLS position, use of repression domains, and position of gRNA target. Our optimal system comprised of dCas12a E925A with a single C-terminal NLS and Mxi1 or MIG1 repression domain enabling a 97% downregulation of a reporter gene. We also extend this system to enable inducible regulation via RNAP II-controlled promoters, demonstrate position-dependent effects in crRNA arrays, and use multiplexed regulation to stringently control a heterologous β-carotene pathway. Together these findings offers valuable insight into the design constraints of dCas12a-based CRISPRi and offers new avenues for flexible and efficient gene regulation in S. cerevisiae.