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

Modification of specific Ser and Thr residues of nucleocytoplasmic proteins with O -GlcNAc, catalyzed by O -GlcNAc 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 -GlcNAcase (OGA), precise functional dissection of site-specific O -GlcNAc modification in vivo is currently not possible without affecting the entire O -GlcNAc proteome. Exploiting the fortuitous promiscuity of OGT, we show that S -GlcNAc is a hydrolytically stable and accurate structural mimic of O -GlcNAc that can be encoded in mammalian systems with CRISPR–Cas9 in an otherwise unperturbed O -GlcNAcome. Using this approach, we target an elusive Ser 405 O -GlcNAc site on OGA, showing that this site-specific modification affects OGA stability.