Generation of P735R-knockin mice. (A) Schematic domain representation of UDP-GlcNAc-2-epimerase and ManNAc kinase in human GNE, and the structure of dimeric kinase domains (PDB ID: 2YHW). (B) Structural prediction of kinase domain carrying C594Y mutation, showing that 1 rotamer causes severe clashes in all 3 rotamers. (C) Structural prediction of kinase domain carrying P735R mutation, showing severe clashes for all the 22 rotamers. Mutagenesis and rotamer selection were performed using PyMOL, in which the gray sticks indicate the mutated residues and the red octagon disks indicate significant van der Waals overlap, meaning atoms are close to other atoms causing clashes. (D) Diagram of the targeted genomic sequence in the Gne^P735R/P735R (hereafter, mt/mt) mouse using the CRISPR-Cas9 system. The synonymous mutation p.Leu737 (CTG to TTA) was introduced as a blocking mutation to prevent re-cutting by Cas9 after homology-directed repair. (E) Illustration of the RFLP-based genotyping assay using PCR and restriction endonuclease NlaIV. (F) Genotyping results visualized using 2% agarose/TBE gel.