Fig. 4: Repression and derepression dynamics of degron-Cas-repressor systems.
From: CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression
a Schematics of the experimental setup to study the dynamics of repression for the different degron-Cas-repressors. dTAG-13 is withdrawn at time 0 and then repressor (tBFP) and target (ESRRB-mCherry) levels are measured by flow cytometry over time. Upon ligand withdrawal, the degron-Cas-repressor level increases to then reach a steady state. We estimate the time required to reach half of the maximal repressor level (t1/2) by fitting an ordinary differential equation (ODE) to the experimental data. To assess whether target repression is immediate upon repressor upregulation (Δt=0, solid line) or occurs with a delay (Δt>0, dashed line), the mCherry data were fitted with an ODE model, where target gene expression varies as a function of time and of repressor concentration with or without assuming a delay between repressor upregulation and target gene repression (Δt). See Supplementary Fig. 4e and the methods section for details on the modelling approach. b Bg-subtr. MFI for repressor (tBFP, scaled between 0 and 1) and target gene (mCherry, rel. to initial time point) for the different degron-Cas-repressor systems, for three biological replicates (dots). The black lines in each plot represent the best fit of the ODE model. For tBFP, the t1/2 ± its standard error is indicated for each degron-repressor system. For mCherry, the continuous line shows the result of the ODE model with Δt = 0 h and the dashed line the ODE model with the Δt that minimises the Mean Absolute Error of the model compared to the experimental measurements. c To study the dynamics of derepression associated with each system, dTAG-13 is added back to the medium after 4 days in the absence of dTAG-13 (in the presence of repression). The same type of model as shown in (a) is used and we estimate the delay between repressor degradation and target gene derepression. d Same as in (b) but for the derepression dynamics experiment schematised in (c). The two KRAB-based repression systems show a substantial delay in target gene derepression.
