Fig. 2

TEV protease-based sensor design. Different designs for TEV protease-based sensors to detect molecular events. GPCR activation sensors are used as examples for Tango, iTango and ChaCha system. Protein–protein interaction sensors are used for SPARK1/2 system. a Tango system: TCS and a transcription factor (TF) are fused to the c-terminal of a GPCR. Upon activation, β-Arrestin tagged with TEVp will bind intracellular loop of GPCR and cleave TCS when exposed to release TF. b iTango system: TF is coupled to the C-terminal of a GPCR via TCS, which is caged by AsLOV2/Jα. Upon activation, β-Arrestin tagged with TEVp-C will bind intracellular loop of GPCR tagged with TEVp-N, which will form functional TEV and cleave TCS when exposed to light stimulation and release TF. c ChaCha system: The TCS and a dCas9 based TF are fused to β-Arrestin while TEVp is fused to the C-terminal of a GPCR. After interaction of β-Arrestin and GPCR upon activation, TCS is cleaved and dCas9 is released. d SPARK1 system: Protein A is fused to the AsLOV2 caged TCS and TF. Protein B is fused to TEVp. Upon interaction between proteins A and B, blue light can dismiss the protection from LOV domain and leave TCS cleaved by TEVp, which then release TF. e SPARK2 system: Similar to SPARK1 design. Instead of using blue light, luciferase is fused to protein B. After interaction between proteins A and B, the luciferase with luciferin activates the LOV domain via BRET, allowing the TEVp to cleave TCS and release TF. f Released TF translocate into nuclear and initiate reporter gene expression. Notably, dCas9 with sgRNA can easily modify endogenous gene expression other than reporter gene