Fig. 1.

CRISPReader drives promoterless gene expression by coupling the transcriptional and translational mechanisms. a CRISPReader was constructed by combining transcriptional and translational platforms. The dCas9-VP64 protein robustly activated transcription of reporter constructs when combined with sgRNA targeting sequences near the TATA box. Then, the RNA activator led to the formation of initiation factor complexes involving eIF4G and recruited ribosomes to initiate translation. b The results of the dual luciferase assay. An unregulated TK promoter-driven gene encoding firefly luciferase was used as a control. Reported data are the mean ± SD from five experiments. **P < 0.01, compared with the sgRNA-negative control using the paired, one-sided t test. c Mechanisms of the CRISPReader designed to drive the gene cluster expression. After dCas9-VP64-mediated transcription, the RNA activators bound to each targeted mRNA and independently initiated mRNA translation. d CRISPReader activated the expression of each ORF. The results of the dual luciferase assay are shown at the top. Reported values are presented as the mean ± SD, and the experiments were repeated five times. **P < 0.01, compared with the negative control using the paired, one-sided t test. The expressions of GFP and RFP were detected by fluorescent microscopy. Representative images of the transfected cells are shown at the bottom. Scale bar 1000 μm