Streamlining and Enhancing Guide RNA Expression for Programmable Large-Scale Multiplex of Desired Genes in Saccharomyces Cerevisiae

While metabolic engineering has successfully led to the renewable production of high-value chemicals such as artemisinin and 1,3-propanediol, it nonetheless suffers from the slow rate of genomic modification. As a solution, the CRISPR-dCas9 system (typically with fused activator or repressor domains) has been used to enable modification of gene expression in a rapid, transferrable manner. This talk covers three major advances we have made in adapting and expanding this system for the yeast Saccharomyces cerevisiae.

First, to bypass the typical binary on/off gene modulation inherent in this system, we previously developed a scheme for graded dCas9 regulation with a large dynamic range of perturbation. We accomplish this by modulating dCas9-activator/repressor proximity relative to the desired gene target. Linking dCas9-mediated graded gene expression with desired phenotypes to Systematically Test Enzyme Perturbation Sensitivities (STEPS) afforded rapid iterative optimization of glycerol and 3-dehydroshikimate titers (5.7 and 7.8-fold improvement, respectively). While this approach was a step forward for more rapid strain engineering, it can solely access a small combinatorial expression space due to the use of repetitive and bulky Pol III expression cassettes for each individual sgRNA.

Second, we thus address the combinatorial optimization challenge by developing a streamlined approach for gene modulation. This approach uses Pol II promoters and ribozymes to express multiple sgRNAs from a single cistron while at the same time re-purposing the dCas9-VPR activator to predictably activate or repress target genes based on its targeting location within the desired gene locus. In doing so, we achieve an unexpected yet beneficial result-that strong Pol II expression of sgRNAs produces nearly 4-fold more mature sgRNA than the traditional Pol III SNR52 promoter, thus affording stronger CRISPRi repression and doubling the dynamic range of dCas9-VPR modulation of the strong TPI1 promoter.

Third, we demonstrate a final streamline of our approach that optimizes the expression cassette for more efficient excision of sgRNAs via endogenous RNA processing. Ultimately, these more optimized elements further enhance dCas9 perturbation efficiency, reduce the DNA load, and facilitate easy cloning schemes for rapid combinatorial assembly of arrays that can modify entire pathways. Using this system, we demonstrate that we can rapidly synthesize and transform a desired, complex genotype into cells and showcase the portability of this approach across multiple different strains. We foresee this approach as a tool to rapidly condense genome regulation into a singular cassette that can be transferred to different strain backgrounds by a singular transformation step.