Fig. 4: Transcriptional regulation and genome editing using iCROP-engineered (d)Cas9 protein or MCP_VP64.

a Schematic representation of the iCROP-modified (d)Cas9 protein. dCas9 or Cas9 protein bearing the HRV protease at the N-terminus and the cognate CS at position R535 is fragmented into two inactive parts, unless the protease inhibitor is present. In the presence of inhibitor, (i) active dCas9 associates with a target sgRNA fused to an MS2 loop, which co-localizes the MCP protein fused to a transcriptional activation domain in the promoter region of the target gene, thereby activating transcription; and (ii) active Cas9 associates with a target sgRNA introducing mutations in a target genomic locus. b SEAP produced by cells constitutively expressing iCROP-dCas9 (P_mPGK-HRVp_ΔQ182-dCas9_CS-R535-pA), MS2 loop fused to an sgRNA targeting the insulin promoter (P_U6-sgRNA(P_INS)-MS2), MCP fused to the transactivation domain (pKK44, P_hCMV-MCP-p65_TA-HSF1_TA-pA) and SEAP expression under an insulin-responsive promoter (P_INS-SEAP-pA), and treated for 24 h with the indicated concentrations of rupintrivir. c Schematic illustration of the iCROP-MCP_VP64 system for dCas9-guided transcription regulation in response to rupintrivir. d Activation of gene expression from the endogenous genomic target with the iCROP-MCP_VP64 system. Cells were co-transfected with dCas9, iCROP-MCP_VP64 and sgRNA targeting the IL-12B promoter (sgRNA(P_IL-12B)-MS2) and treated with the specified rupintrivir concentrations for 48 h before analyzing IL-12 levels in the supernatant by ELISA. e Kinetics of iCROP-MCP_VP64-based activated IL-12 expression. Time-course analysis of IL-12 levels in culture supernatants from cells treated either with 1 µM rupintrivir or vehicle. f Mutation rate in the EMX1 gene in cells expressing iCROP-Cas9 and sgRNA(EMX1), and treated with indicated concentrations of rupintrivir for 48 h before NGS analysis. Cells co-transfected with non-modified Cas9 and sgRNA(EMX1) were included as a positive control. g Heat-map representing time- and dose-dependent EMX1 relative indel frequency for iCROP-Cas9. Relative indel frequency was calculated as the ratio between the mutation rate at each specified dose-time point and the maximal mutation rate (n = 3 biological replicates). h Frequency of indel mutations in the genomic loci of VEGFA, TNFRSF1A and ACE2 genes in cells co-transfected with iCROP-Cas9 and each sgRNA targeting gene, and kept for 48 h in the absence or presence of rupintrivir (1 μM) (sgRNA variants with the highest fold induction of mutation rates are represented (see Supplementary Fig. S12g for efficiency of other sgRNA). In panels b, d, e, f, h, data are shown as mean ± SD (n = 3 biological replicates). Statistical significance was calculated by means of Welch’s two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.