Fig. 2

Further characterization of drug-inducible CRISPR platforms and benchmarking against literature designs. (a) Time-course of EGFP disruption activity after drug treatment. MC-38 cells with stable Cas9 expression were transduced with constitutive (grey lines), DOX-inducible (light and dark blue lines) and IPTG-inducible (light and dark red lines) sgRNA expression vectors and selected with puromycin. Parental MC-38 cells were transduced with an EGFP reporter as a control (green lines). 1 μg/mL DOX or 1 mM IPTG was used to induce the sgRNA expression. (b) RT-qPCR analysis of sgRNA levels in MC-38 cells. Chemical inducers were applied to cell culture medium on Day 0 and washed out on Day 2. 1 μg/mL DOX or 1 mM IPTG was used to induce the sgRNA expression. (c) RT-qPCR analysis of sgRNA levels with or without DOX treatment. 1 μg/mL DOX was used to induce the sgRNA expression. Data represent mean ± SD (n = 3). P values were derived from t tests: ***P < 0.001. (d) Comparison of background activity and inducible efficiency with existing (DD-Cas9 and DFHR.Cas9-DHFR) designs using the EGFP disruption assay. Induction conditions: 1 μg/mL DOX for 2xTetO system, 1 mM IPTG for 2xLacO system, 1 μM Shield-1 for DD-Cas9, and 10 μM TMP for DHFR.Cas9.DFHR. Data represent mean ± SD (n = 3). P values were derived from t tests: ***P < 0.001