Fig. 1: Fundamental characterization of Tl-CRISPRi in the E. coli K-12 MG1655.

a A scheme of dCas13-based translation repression. The dCas13 protein and its cognate guide RNA are assembled, forming a RNP binary complex. We redirected it to bind to the translation initiation site of specific mRNA to block the translation of the ribosome and turn off the expression of a target gene. b Adopting the Tl-CRISPRi for three different reporter genes (mCherry, GFP, nanoluc) with six different spacers for each gene. NT represents the strain with the non-target guide RNA (RfxgRNA-NT). c, d Simultaneous and specific reporter gene knockdown by the Tl-CRISPRi. Each guide RNA with the effective spacer (mCherry sp1, GFP sp1, nanoluc sp1) specifically down-regulated the target gene. Simultaneous gene knockdown was achieved by introducing multiple guide RNAs. The relative expression (b–d) was determined based on the specific fluorescence or luminescence level (RFU or RLU/OD₆₀₀) and normalized by setting the value of the NT strain as 100%. The error bar represents the mean ± standard deviation from the biologically independent cell cultures (n = 3), and the white dots indicate the actual data points. The P-value of each strain’s dataset was determined by the two-tailed Student’s t-test compared to the dataset of the NT strain. The asterisk above the bar indicates the P-value. NS not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a source data file.