Fig. 2: Comparing the Tx-CRISPRi and the Tl-CRISPRi on regulating the polycistronic gene expression (mCherry-GFP).

The reporter gene operon consisting of mCherry and GFP was expressed from the chromosome of E. coli. a, b The expected difference between transcription-level control and translation-level control toward polycistronic gene expression. If the dCas9 blocks the upstream gene in the operon, all downstream genes are simultaneously turned off, called the polar effect. We reasoned that dCas13-based translation-level knockdown on the upstream gene would not intrinsically down-regulate the expression of downstream genes in the operon if each cistron is independently translated from its ribosome binding site. c–f The relative expression level when each reporter gene was targeted by the Tx- or Tl-CRISPRi. For the Tl-CRISPRi, the spacer mCherry sp1 or GFP sp1 was applied to knock down the expression of mCherry or GFP, respectively. The relative expression was determined based on the specific fluorescence level (RFU/OD₆₀₀) and normalized by setting the value of the NT (non-targeted) 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. Source data are provided as a source data file.