Figure 5. Activation of biosensors in mammalian cells and regulation of CRISPR/Cas9 activity.
(a) Concentration dependence of response to digoxin for constructs containing digoxin TF-biosensors and Gal4 UAS-E1b-EGFP reporter individually integrated into K562 cells. GR60S,L77F-PRO1-V serves as a digoxin insensitive control. (b) Concentration dependence of response to progesterone for constructs containing progesterone TF-biosensors and Gal4 UAS-E1b-EGFP reporter individually integrated into K562 cells. GR60S-DIG1-V serves as a progesterone insensitive control. (c) Time dependence of response to 100 nM digoxin for constructs containing digoxin TF-biosensors and Gal4 UAS-E1b-EGFP reporter individually integrated into K562 cells. GR60S,L77F-PRO1-V serves as a digoxin insensitive control. (d) Time dependence of response to 25 μM progesterone for constructs containing progesterone TF-biosensors and Gal4 UAS-E1b-EGFP reporter individually integrated into K562 cells. GR60S-DIG1-V serves as a progesterone insensitive control. (e) DIG3 and PRO1 fused to the N-terminus of S. pyogenes Cas9 were integrated into a K562 cell line containing a broken EGFP. EGFP function is restored upon transfection of a guide RNA and donor oligonucleotide with matching sequence in the presence of active Cas9. The data are presented as mean fluorescence ± s.e.m. of three biological replicates.