Fig. 3: Assessing tunability of degron-Cas-repressor systems.
From: CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression
a Experimental design: degron-Cas-repressor cell lines expressing ESRRB-mCherry are transferred from medium containing high concentration of dTAG-13 (repressor degraded) to media with a range of dTAG-13 concentrations. The degron-Cas-repressor levels are expected to decrease with increasing dTAG-13 concentrations, resulting in a rise in target gene expression (mCherry). Quantification of ESRRB-mCherry and degron-Cas-repressor (tBFP) levels after 2 and 4 days by flow cytometry will then allow the distinction between analog (homogenous intermediate levels) and digital (a mixture of positive and negative cells) repression. b, c Density plots of tBFP (b) and mCherry (c) expression levels measured by flow cytometry after 4 days of treatment. One biological replicate is shown. dCas9 and KRAB-Split-dCas9 are the same construct (KRAB-Split-dCas9) with 100 µM ABA being added to the KRAB-Split-dCas9 4 days before the measurement. The dotted line shows the 99th percentile of non-fluorescent control cells. d tBFP (top) and mCherry (bottom) levels normalised to cells expressing NTC guides. The mean of three biological replicates (dots) ± standard deviation (vertical bars) is shown. bg-subtr. = background-subtracted; MFI = Median Fluorescence Intensity (e), mCherry MFI at different doses of repressor (tBFP MFI) after 4 days of titration. The mean of three biological replicates is shown. f Normalised 2D density plots showing tBFP and mCherry levels in populations of cells treated with different dTAG-13 concentrations (indicated on the right). A.F.U. = Arbitrary Fluorescence Units.
