Fig 1: Overview of variant enzymatic function characterization using SMuRF.a, Functions of most α-DG glycosylation enzymes can be evaluated by the IIH6C4 antibody. Blue texts mark the enzymes involved in the glycosylation of α-DG Core M3 and its extension. Bold arrows link enzymes to the modifications or glycan additions they catalyze; for instance, POMK catalyzes mannose phosphorylation. Dol-P-Man is dolichol phosphate mannose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Rbo5P, ribitol-5-phosphate; Xyl, xylose; GlcA, glucronic acid. b, A universal workflow of SMuRF. SMuRF accompanies saturation mutagenesis with functional assays. Here, saturation mutagenesis is achieved by delivering variant lentiviral particles to the engineered HAP1 platform where the endogenous gene of interest (GOI) was knocked-out and stable DAG1 overexpression was established through lentiviral integration. A fluorescence-activated cell sorting (FACS) assay was employed to separate the high-function population and the low-function population.