Gene Regulation with O-GlcNAc Glycosylation by dCas9-OGT/OGA Fusion Proteins

Abstract

O-linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification plays important roles in cellular network/diseases such as autophagy, transcriptional activity, protein stability, phosphorylation modification competition, cancers, neurodegenerative disorders, etc. However, there are still lots of unknown roles of it in gene regulation especially while there is existence of more than 1000 transcription factors and many other coactivators/repressors which might have influence in expression level with or without O-GlcNAc modification. In this project, we use CRISPR technology with catalytically inactive Cas9 from Streptococcus pyogenes fused with the only writer and eraser of O-GlcNAc to selectively target to the DNA sequence of interest by guide RNA to see what the result of additional modification or cleavage of O-GlcNAc on proximal proteins in gene regulation is. A cost and time efficient way for guide RNA construction is developed in the project with one-piece PCR and Gibson assembly for 2 different backbones of guide RNA: 11 guide RNA for each. Other different cloning methods have also been used for the future work. For the future work, second reporter will be introduced to normalize the luciferase signal. In addition, new metabolic O-GlcNAc reporter and Y289L GalT/ UDP-GalNAz could be used to find out proteins which might be modify by dCas9-OGT/OGA with click reaction. Furthermore, split OGA/Turbo ID system could also be used to reduce the background and find out proteins/modification sites which might be important to the gene regulation for DNA-protein or protein-protein interaction.