Figure 4.

Inducible degradation systems to control CRISPR-Cas9 genome editing

(A) Auxin-induced degron (AID) system.⁶⁸ dCas9 can be fused with the degron IAA17. T1R1 is the auxin-binding receptor from the rice plant, and auxin is a plant hormone. In non-plant cells, T1R1 complexes with the conserved ubiquitin E3 ligase and Skp1-Cul1-F (SFC) box. dCas9 has been shown to be degraded with the AID system but has not been demonstrated with the Cas9 nuclease. (B) Degradation tag (dTAG) system.⁸⁸ Cas9 can be fused to multiple mutant FKBP12 (i.e. FKBP12^F36V). Once FKBP12^F36V is polyubiquitinated, Cas9 will be degraded along with the FKBP12^F36V. (C) Small molecule-controlled Cas9 repressible system.⁸⁷ Cas9 is fused to a SMASh tag. Upon the addition of the inhibitor asunaprevir (ASV) that attaches to the NS3 protease and NS4A degron, Cas9 is degraded. When asunaprevir is removed, the NS3 protease self-cleaves and is degraded with the NS4A, while Cas9 is fully functional.