Fig. 5

Gene editing of SHOX promoter G4s restores SHOX expression and chondrogenicity in WRN^−/− MSC. a Schematic illustration of sgRNAs targeting two G4 sites in SHOX promoter. Red: G nucleotides. Box: G > C mutants tested in luciferase assay in Fig. 2c. Indel percentage of sgRNA targeted MSC is calculated by (1- non-indel%) predicted by TIDER. b Averaged fold change of SHOX mRNA in G4 gene-edited WRN^−/− MSC as compared to scramble sgRNA. Error bar: SEM. N = 3. t-test confidence (1-tailed, unequal variance) against WRN^−/−: *p < 0.05, **p < 0.005. Western blot confirms elevated SHOX protein level in G4 gene-edited MSC. Number represents relative SHOX protein against WRN^−/− MSC calculated by intensity of SHOX/ACTININ. c Alcian blue and immunofluorescent staining of SOX9, COL2 and SHOX in D10 MSC-differentiated chondrogenic pellets. N = 3. Scale bar: 100 µm. d qPCR analysis showing gene expressions of SOX9, COL2, SHOX and WRN relative to GAPDH (dCT) in G4 gene-edited MSC-differentiated chondrocytes at D0 and D10. N = 3. t-test confidence (1-tailed, unequal variance) of D10 chondrocytes against WRN^−/−: *p < 0.05, **p < 0.005