Genetic recoding to dissect the roles of site-specific protein O-GIcNAcylation

Modification of specific Ser and Thr residues of nucleocytoplasmic proteins with O-GIcNAc, catalyzed by O-GIcNAc transferase (OGT), is an abundant posttranslational event essential for proper animal development and is dysregulated in various diseases. Due to the rapid concurrent removal by the single O-GIcNAcase (OGA), precise functional dissection of site-specific O-GIcNAc modification in vivo is currently not possible without affecting the entire O-GIcNAc proteome. Exploiting the fortuitous promiscuity of OGT, we show that S-GIcNAc is a hydrolytically stable and accurate structural mimic of O-GIcNAc that can be encoded in mammalian systems with CRISPR-Cas9 in an otherwise unperturbed O-GIcNAcome. Using this approach, we target an elusive Ser 405 O-GIcNAc site on OGA, showing that this site-specific modification affects OGA stability.