A Toolkit of Inducible Degron-Tagged dCas9 Effectors for Multi-Directional Drug-Tunable Control of Synthetic Biology Applications

CRISPR/dCas9 based technologies are enabling the artificial control of gene transcription, a capability that will be highly beneficial to the advancement of our understanding of natural gene regulatory mechanisms and for the design of synthetic control networks that bestow cells with desired properties for synthetic biology and biotechnological applications. The nuclease-deactivated variant of CRISPR/Cas9 proteins (dCas9) fused to heterologous transactivation domains can act as a potent inducer of gene expression in mammalian cells. A distinct advantage of dCas9 over other artificial activators such as ZFNs and TALE-TFs is that expression of a single dCas9 construct per cell is sufficient, whose activity can be directed to multiple specific sites on the DNA via sequence-directed guide RNAs, with their typically rapid turn-over time. In such a system the long-term presence of a stable dCas9 effector can be a draw-back precluding the ability to switch rapidly between repressed and activated states of target gene expression and imposing a static environment on the synthetic regulatory circuits in the cell. To address this issue we have generated a toolkit of conditionally degradable or stabilisable dCas9 proteins. We show that fusion of the Auxin-inducible degron (AID) to dCas9 generates a rapidly degradable DNA-binding factor, while fusion with the ecDHFR destabilisation domain (ecDDD) produces a protein that is stabilised by drug addition. We extend the system by investigating the potential of orthologous Cas9/Cpf1 proteins to be controlled in a similar manner, thus opening options for multidimensional control of functional activities through combinations of orthologous drug-tunable artificial transcription factors. Finally, we have adapted the MS2 aptamer-containing sgRNA dependent CRISPR/dCas9 set-up (SAM system) by placing the ecDHFR degron on the MCP-VP64 effector module, which, in combination with AID-dCas9, creates a bi-directionally controllable artificial DNA-binding factor that allows for precise fine-tuning of functional activity.