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. Author manuscript; available in PMC: 2016 Nov 2.
Published in final edited form as: Nat Biotechnol. 2016 May 2;34(6):646–651. doi: 10.1038/nbt.3528

Figure 2. Creation of a switch-like Cas9 though insertion of the Estrogen Receptor ligand binding domain.

Figure 2

(a) Schematic of the screen / counter-screen procedure to select for ligand responsive Estrogen Receptor ligand binding domain (ER-Cas9) insertions. (b) Dose-response curve to 4-HT. darC9:231 has an IC50 of 440 ± 70 nM (S.D) and a Hill coefficient of 1.04 as expected for non-cooperative binding of 4-HT to ER-LBD. (c) Single cell analysis of darC9:231 binding in response to increasing concentrations of 4-HT. Flow cytometry data tracks ensemble data demonstrating a > 9 fold switch between darC9 based GFP repression plus and minus 4-HT (darC9 GFP signal without 4-HT mean: 24,310 (au) and with 100 µM 4-HT: 2,631 (au) (d) Dose response of darC9:231 binding to various ligands (B–E and DES are beta-estradiol & diethylstilbestrol). Response is normalized to vector control fluorescence under the same conditions. (e) Switching of arC9:231 cleavage activity. Transformation assays demonstrate that ligand dependent arC9 switching also extends to cleavage activity, t-test (** p-value <0.01). All experiments performed in E. coli.