CRISPR-Cas9 Gene Editing of Saccharomyces cerevisiae to Study Membrane Traffic

The objective of this study was to determine how active transporters, located at the plasma membrane of a cell, are regulated. This study had a specific focus on regulated endocytosis in the model system Saccharomyces cerevisiae (yeast). Endocytosis in S. cerevisiae is initiated once membrane proteins are ubiquitinated. This ubiquitin tag allows the cell to properly select membrane transporters and place them into endocytic vesicles. Ubiquitination is determined by ubiquitin ligase, Rsp5 in yeast, and its adaptors. The adaptors involved in this research include arrestins, Art4, Bul1, and Bul2, and are believed to make a molecular interaction with signals on the transporter that form a lock and key interaction. The overall goal of this study is to use random mutants and genetic screens to identify critical amino acids used on both the transporter and arrestin-adaptor pairs that contribute to this lock and key model. This study focused on the use of the CRISPR-Cas9 genetic engineering system which allowed the preparation of yeast strains for genetic screens by deletion of the endogenous pump JEN1 and adaptors ART4, BUL1, and BUL2. Two parts of the CRISPR strategy consisted of first generation of a guide RNA plasmid by in vitro phosphorylation and ligation of a DNA oligonucleotide, followed by transformation of guide RNA and CAS9 genes into yeast cells. Second a repair template for homology directed repair was generated by overlap PCR. Upon cutting of the genome by Cas9 + guide RNA cellular homologous repair pathways will repair the double strand break. Our repair template will cause the repaired DNA region to have a targeted deletion. Only repairs that incorporate this deletion will escape further Cas9 cutting and survive. Future directions include sequencing and validation of the mutants, which will then enable genetic screens to detect the arrestin-transporter interaction mechanism.