Fig. 7: Titrating Oct4 with CasTuner reveals a threshold level to induce the trophectoderm transcriptional program.
From: CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression
a–c Oct4 titration in mESCs (1.8XX Nanog-mCherry cell line expressing FKBP12F36V-hHDAC4-dCas9) for 3 days. a Oct4 expression fold change at different dTAG-13 concentrations as measured by qRT-PCR, for 3 biological replicates (small dots). Vertical lines indicate the standard deviation and larger dots the mean. b Heatmap of average expression fold change at different dTAG-13 concentrations relative to the 500 nM dTAG-13 condition for the indicated lineage markers. mESCs = mouse embryonic stem cells; EpiSCs = epiblast-like stem cells; PE = primitive endoderm; TE = trophectoderm. A two-sided t-test between each dTAG-13 condition to the 500 nM dTAG-13 condition was used. Asterisks indicate p value < 0.05. Exact p values are given in Source Data. c Dose-response relationship between Oct4 and target gene levels measured by qRT-PCR, shown as the mean of three biological replicates (dots). Horizontal and vertical lines indicate the standard deviation for Oct4 and its target genes, respectively. The black curve represents the most likely Hill curve fitted using a non-linear least square approach (see Methods). For all shown target genes, except Nanog, the red vertical line marks the fold change of Oct4 that leads to half of the maximal expected fold change variation of the target ([O]½), according to the fitted model, which is also reported in each plot, together with the Hill coefficient n. The R2 for the fits ranges between 0.92 and 0.99. The Oct4-Nanog dose-response relationship is non-monotonic and a dashed black line is used to connect the mean of the different average measurements.
