Fig. 5.

Stereo-tuner dCas9 system. a Schematic overview of the system. An effector-less dCas9 is tagged with the auxin-inducible degron allowing inducible degradation upon addition of auxin. Two MS2 aptamer sequences are included in the single-guide RNA scaffold, as used in the Crispr/SAM system⁸. A transcriptional effector domain (e.g., VP64) is brought in by the MS2 aptamer-interacting MS2 core protein (MCP) which is fused to the ecDHFR destabilisation domain (ecDDD). Addition of the small molecule drug trimethoprim (TMP) improves the stability of naturally destabilised ecDHFR fusion proteins. PIC, Polymerase II initiation complex. b Western blot with HA antibody showing TMP concentration dependent stabilisation of the ecDHFR-MCP-VP64-5HA protein. β-actin antibody is included as loading control. c Dual-luciferase assay using a firefly luciferase reporter driven from a minimal promoter with eight copies of a guide RNA target sequence (gRNA-1) in an effector-less AID-dCas9 expressing cell line. A ubiquitous Renilla luciferase construct was co-transfected as internal control. In the presence of the MS2 aptamer bearing gRNA-1, luciferase activity is produced by transfection of a dCas9-VP64 plasmid (first lane) or by the co-expression of the endogenous AID-dCas9 with the pecDHFR-MCP-VP64 effector module, and can be enhanced by TMP administration. Addition of auxin to the medium, affecting IAA17-dCas9 stability, abrogates luciferase activation. Grey columns, controls; Light blue column, no TMP, no auxin; Dark blue columns, TMP added; Red columns, auxin added. Error bars denote ± s.d. (n = 3) (two-tailed t-test, (**) P < 0.01)