CRISPR-Cas RNA Scaffolds for Transcriptional Programming in Yeast

CRISPR scaffold RNAs (scRNAs) provide a modular system for locus-specific transcriptional programming. scRNAs are generated by extending CRISPR guide RNA sequences with domains that recruit RNA-binding proteins, thus physically linking DNA binding and protein recruitment activities. A single scRNA molecule encodes information about the target locus and instructions about what regulatory function to execute at that locus. Sets of scRNA constructs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. Such programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point, and this approach has been recently applied to flexibly redirect flux through a complex branched metabolic pathway in yeast. This protocol describes how to construct multi-scRNA transcriptional programs in yeast, including target site selection, cloning strategies, and yeast engineering.