Fig. 2: Design and implementation of the cMSC.

a Schematic showing synthetic P_aMYC expressing GFP in MYC^low and MYC^high cells. P_aMYC exhibited low transcriptional activity in MYC^low cells but high activity in MYC^high cells. b Schematic illustration of synthetic P_rMYC expressing mCherry across c-MYC expression gradients. P_rMYC exhibited high transcriptional activity in MYC^low cells but low activity in MYC^high cells. c Mean fluorescence intensity (MFI) of GFP- and mCherry-driven expression of P_aMYC and P_rMYC in BFP⁺ cells, respectively, over a range of siRNA and tetracycline concentrations. The data are presented as the mean ± SD, n = 3 individual experiments. d Schematic showing the composition of the preliminary cMSC and its working mechanism in MYC^low and MYC^high cells. The preliminary cMSC consisted of two parts. The first part was P_aMYC, which expresses the GOI expression cassette whose 3’-UTR contains ten sequences of split-ribozyme-1 (split-1) in tandem. The second part consisted of P_rMYC expressing split-ribozyme-2 (split-2). The working mechanisms of the preliminary cMSC in cells are as follows: In MYC^low cells (left), low expression of c-MYC results in low activity of P_aMYC and high activity of P_rMYC. In step 1, P_aMYC resulted in weak mRNA (GFP-10×split-1) transcription, whereas P_rMYC exhibited strong transcription of split-2. In step 2, a large amount of split-2 would interact with GFP-split-1, leading to the degradation of GFP mRNA through the ribozyme-based mRNA degradation process, resulting in almost no expression of GFP (step 3). In MYC^high cells (right), high expression of c-MYC resulted in high activity of P_aMYC and low activity of P_rMYC. P_aMYC resulted in strong mRNA (GFP-10×split-1) transcription, whereas P_rMYC resulted in weak transcription of split-2. In step 2, a small amount of split-2 would partly interact with GFP-split-1, leading to only partial degradation of the GFP mRNA, which led to a small amount of GFP translation (step 3). e Schematic showing the composition of the optimized cMSC and its working mechanism in MYC^low and MYC^high cells. The optimized cMSC consists of two parts. First, 6 binding sites of KLFa (6×KLFa-BS) were inserted upstream of P_aMYC. The reengineered P_aMYC (containing KLFa-BS) was used to drive the expression of a 5’ cassette encoding GFP, a T2A coding sequence, a cassette encoding KLFa and 3’ split-ribozyme-1 in tandem (GFP-T2A-KLFa-10×split-1). In the other part, P_rMYC was used to express split-ribozyme-2 (split-2). The working mechanisms of the optimized cMSC in cells are as follows: In MYC^low cells (left), low expression of c-MYC results in low activity of P_aMYC and high activity of P_rMYC. In step 1, P_aMYC resulted in weak mRNA (GFP-T2A-KLFa-10×split-1) transcription, whereas P_rMYC exhibited strong transcription of split-2. In step 2, a large amount of split-2 would interact with the mRNA of GFP-T2A-KLFa-10×split-1, leading to the degradation of GFP-T2A-KLFa-10×split-1 mRNA through the ribozyme-based mRNA degradation process, resulting in almost no expression of GFP (step 3). In MYC^high cells, high expression of c-MYC resulted in high activity of P_aMYC and low activity of P_rMYC. In step 1, a large amount of GFP-T2A-KLFa-10×split-1 is transcribed by P_aMYC, whereas a small amount of split-2 is produced by P_rMYC. In step 2, some GFP-T2A-KLFa-10×split-1 mRNAs interacted with split-2 and were degraded, whereas others were retained for the next step of translation. In step 3, small amounts of GFP and KLFa were translated. In step 4, the successfully translated KLFa is transferred to the nucleus, where it binds to the KLFa-BS upstream of P_aMYC, reactivating it (step 5) to transcribe more GFP-T2A-KLFa-10×split-1 mRNA (step 6) and translation (step 7). This positive feedback loop would generate an increasing amount of GFP-T2A-KLFa-10×split-1 mRNAs so that the degradation effect of split-2 transcribed by P_rMYC on GFP-T2A-KLFa-10×split-1 mRNA was negligible in this scenario. f Determining the mean fluorescence intensity (MFI) of GFP-driven expression by the preliminary cMSC and the optimized cMSC in BFP⁺ cells over a range of siRNA and tetracycline concentrations. The data were presented as the mean ± SD, n = 3 individual experiments.