Fig. 4

Inhibition of OGT and OGA by the CRISPR/Cas9 system and their effects on cellular O-GlcNAcylation in K562 cells. A Schematic illustration of CRISPR/Cas9-mediated repression of MGEA5 (encoding OGA) and OGT. The oligo sequences of sgRNAs that specifically recognized the target DNA region are shown. B Western blot analysis of OGA, OGT, and O-GlcNAc levels showing knockdown efficiency and manipulation of O-GlcNAcylation in OGAi and OGTi cells in comparison with CRISPR control (pLenti) cells. Blots were reprobed with anti-β-actin antibody to establish a loading control. (C, D) (left) DNA sequencing spanning over the Cas9 cut site on MGEA5 (C) and OGT (D) from the CRISPR control (pLenti) cells in comparison with the OGAi (C) and OGTi D cells. The guide sequence is underlined in black, and the PAM sequence is denoted by a dotted red underline in the control sample. Vertical dotted lines denote the expected cut site. (right) INDEL spectrum and ICE editing efficiency (Edit eff) determined by ICE software. (lower) The inferred sequences of MGEA5 present in OGAi C and of OGT present in OGTi D cells and their relative proportions (%INDEL) were listed along with the translated amino acids