CRISPRi for elucidating stress regulation in yeast

Research Project, 2019 – 2023

Phenotypic traits are affected by an interplay of genes, thus redirecting cellular fluxes may require alteration of the expression of many genes. Our aim is to gain novel insight in how the regulatory cascades of a cell influence its phenotypic traits. We will alter the expression of many genes involved in stress response of Saccharomyces cerevisiae through the CRISPRi technology. In CRISPRi, an RNA guided protein, dCas9, fused to an activator or repressor is targeted to a specific locus by a co-expressed RNA. By targeting a dCas9-fusion to a promoter, the expression of a gene is gradually altered, while still allowing native regulation. The generated CRISPRi strain library will be screened for increased inhibitor tolerance, a key feature of industrial production hosts. Transcriptomic analysis will be done on selected strains for identifying changes in transcription leading to increased tolerance. A thorough understanding of cellular regulation is needed for the development of cell factories for biochemical conversion of renewable carbon.