Fig. 4. Dual function of Cas endoRNases in the PERSIST platform.

a A tagBFP-encoding OFF-switch reporter (blue line) and an EYFP-encoding ON-switch reporter (yellow line) can be acted on simultaneously by Csy4 in the same cell. The ON/OFF-switch reporters and Csy4 plasmid were co-transfected with plasmids encoding constitutive iRFP720 and mKO2 respectively in order to track their corresponding transfection efficiencies. Summary values were calculated for iRFP720-positive cells. b, A coherent feed-forward loop with dual function Cas protein improves the response of the PERSIST-ON-switch motif. For plasmid ratio calculations, CasE plasmid was maintained at 75 ng. Transfection efficiency of the EYFP reporter and intermediate Csy4 plasmid was tracked by a plasmid encoding constitutive tagBFP while CasE transfection efficiency was tracked by a plasmid encoding constitutive mKO2. Summary values were calculated for tagBFP-positive cells. Fold change (inset) was calculated by normalizing to the EYFP value at the lowest mKO2 bin (see Supplementary Fig. 16) and evaluated at mKO2 = 10,000. Over 1000-fold dynamic range is achieved for certain Csy:CasE ratios compared to roughly 100-fold dynamic range achieved by the ON-switch alone (gray). a, b n = 2 biologically independent samples where each sample represents the evaluation of >1000 transfected cells (HEK293FT). The shaded region is the maximum interquartile range of all cells in each bin.