Controlling Transcription in the Yeast Yarrowia Lipolytica

The yeast Yarrowia lipolytica has been the subject of extensive metabolic engineering and synthetic biology studies due to its ability to make and store intracellular lipids. Recent engineering efforts have been aided by the development of a suite of different engineering tools, including well characterized promoters for heterologous gene expression, a Cre-lox recombinase system for selectable marker recycling, and CRISPR-Cas9-based tools for gene disruptions, integrations, and transcriptional repression. We have adapted our previously developed CRISPR-Cas9 system to enable the activation of transcription (CRISPRa). Different activation domains were systematically compared for their ability to drive expression of GFP from a model synthetic promoter, with the VPR domain giving the highest expression. A series of sgRNAs targeting the same model synthetic promoter were then designed, and co-expressed with a dCas9-VPR fusion protein. Fluorescence was measured, and CRISPRa activation of GFP was observed. The CRISPRa system was then tested on three different native Y. lipolytica promoters expressing GFP, and fluorescence was measured. This CRISPRa system will enable the activation of arbitrary native Y. lipolytica genes to facilitate the production of oleochemicals of interest.