Transcriptional Reprogramming in Yeast Using dCas9 and Combinatorial gRNA Strategies

Transcriptional regulation is a fundamental and vital process occurring in living organisms. Synthetic biology tools for orthogonal control of transcription have been addressed in a large number of studies to harness this virtue of life for increased metabolic flux and product yield. Extending from previous efforts, the nuclease-deficient Cas9 (dCas9) protrudes as a recent and flexible tool towards genome reprogramming. We approached dCas9-mediated transcriptional reprogramming in Saccharomyces cerevisiae using scaffold-RNAs to recruit transcriptional effectors [1] that were subsequently targeted to genes controlling production of isoprenoids in yeast. Our investigations included >100 guide-RNAs (gRNAs) on 14 different yeast promoters. Through multiplex reprogramming we were able to perturb fluxes in targeted pathways based on phenotypic screening and quantitative measurements.

However, the study also highlighted issues in using dCas9 for transcriptional reprogramming in yeast; most of our gRNA library did not significantly de- or increase expression levels in our reporter assay, where GFP was directly fused to each promoter under investigation. We tested multiple gRNAs per promoter and investigated different features such as protospacer adjacent motif distances to transcriptional start sites.

Taken together, this study presented multiplex designs that can significantly perturb yeast isoprenoid production by using dCas9 and scaffold-RNAs for transcriptional reprogramming. This study also contributed perspective to transcriptional reprogramming using dCas9 in baker’s yeast, and our findings indicate that basic understanding of underlying mechanisms require further exploration, before genome reprogramming can be fully predictive and applied in yeast.

Reference

1. Zalatan JG, Lee ME, Almeida R, Gilbert LA, Whitehead EH, La Russa M, et al. Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell. 2014;160:339–50. doi:10.1016/j.cell.2014.11.052.