Fig. 1: Comparison of degron domains to control dCas9 stability.
From: CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression
a Schematic of PiggyBac (PB) plasmids used to assess degron functionality. dCas9 is fused to tagRFP-T (tRFP), followed by a P2A peptide and tagBFP (tBFP) and expressed under the control of the EF1-alpha promoter. A degron domain is fused N-terminally to dCas9, while in the “no-degron control” dCas9 is not degron-tagged. The P2A peptide allows cleavage of (degron-)dCas9-tRFP and tBFP into two separate proteins. The PB plasmid is cotransfected with a hyperactive PB transposase (PBase) into mESCs (Tx1072 line). Cells that have genomically integrated the PB plasmid in (possibly multiple) genomic locations are selected with blasticidin and then sorted by FACS based on their tBFP level with identical gates for all constructs, yielding a homogenous population of cells. b Schematic overview of degron-dCas9 comparison as shown in (c–g). Top: overview over the tested degrons, their ligands (orange) and whether they are destabilised (default stable, blue) or stabilised (default unstable, green) by ligand addition. Bottom: based on the tRFP/tBFP ratios measured without ligand, at maximal ligand concentration and for the no-degron control, three parameters (see Methods for details) are estimated to characterise the ability to control dCas9 for each degron: degradation leakiness (as measure of the minimal destabilisation conferred by the degron), the degradation efficiency (maximal destabilisation) and the dynamic range (maximal fold change). c Density plots for tRFP (top), tBFP (middle) and the tRFP/tBFP ratio (bottom) for one biological replicate after 24 h of treatment. Dotted lines show the 99th percentile of non-fluorescent control cells. A.F.U. = Arbitrary Fluorescence Units. d tRFP-to-tBFP ratio, calculated as Median Fluorescence Intensity (MFI) after subtraction of the background fluorescence. Dots represent the mean of three biological replicates and are connected by lines; vertical lines indicate the s.d. e–g Bar plots of mean degradation leakiness (e), degradation efficiency (f) and dynamic range (g) calculated for the different degrons as depicted in (b). Degron domains from top to bottom are ranked from best to worst for each property. Black dots are single replicates. Error bars indicate the s.d. of three biological replicates. bg-subtr. = background-subtracted.
