Fig. 4: Development and application of tunable Tl-CRISPRi based on the handle-engineered guide RNAs.

a Genetic components for the validation of tunable Tl-CRISPRi system. The DR sequence was mutated to modulate the interaction between the dCas13 and the guide RNA. This tunable Tl-CRISPRi was directed to inhibit the translation of mCherry mRNA in diverse levels, leading to the tunable expression of mCherry. b The secondary structure of the guide RNA handle. This structure was divided into bulge, stem, and flanking sequence. Each part’s bases were mutated, creating candidate DR sequences for the attenuated guide RNA. c The resulting expression levels of mCherry derived from the guide RNAs harboring diverse DR sequences are shown in the graph. d Relative GFP and nanoluciferase expression level against the mCherry while using the guide RNA with the same mutated DR sequence. GFP sp2 and nanoluc sp1 were each adopted for the knockdown of GFP and nanoluciferase as a spacer sequence. All of the reporter genes were expressed from the chromosome of E. coli. e The relative expression level of mCherry in the V. natriegens compared to that of E. coli when the same attenuated guide RNAs were adopted. The Spearman’s correlation coefficient (ρ) and the related P-value determined by the two-tailed Student’s t-test are annotated in each graph (d, e). 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.