Fig. 4: Multi-gene expression can be rapidly tuned using combinatorial CRISPRa programs.

a Combinatorial library encoding all combinations of four CRISPR-activated expression levels across three genes. The library expresses three scRNAs (variants of J306, J506, and J606). Each scRNA is present in the library in three truncation variants to generate high, medium and low levels of expression of their target promoters (J3, J5, and J6, respectively). In addition to the three truncation variants, the library contains strains with an off-target scRNA in place of each of the J306, J506, and J606 scRNAs to encode a condition in which the target promoter remains unactivated. The lengths of the J306 scRNA variants are 20, 14, and 11 bases. The J506 scRNA variants are 20, 18, and 14 bases. The J606 scRNA variants are 20, 18, and 17 bases. b Use of the combinatorial scRNA library to specify the expression of multiple genes independently. Each member of the combinatorial scRNA library was delivered to a strain harboring a plasmid expressing J3-gfp, J5-bfp, and J6-rfp reporters, generating 64 strains expressing different combinations of the three fluorescent proteins. Points represent the flow cytometry median of GFP, BFP, and RFP from each strain, normalized to the average of the maximum strain across the experiment. The heatmap table below the plot indicates the encoded promoter expression for each strain, as described on the bottom right. Dashed lines represent the Relative Fluorescence/OD₆₀₀ of strains harboring only one of the three fluorescent reporters and only the cognate scRNA (tested with RFP output; see Supplementary Data 2 for plasmids and Supplementary Fig. 9a for variation in single-channel expression), again normalized to the maximum value. Bars in panel b represent the average ± standard deviation calculated from n = 3 biologically independent samples, except strain #9, for which only n = 2 samples grew successfully. The sequence of each scRNA in the combinatorial library can be found in Supplementary Data 1. The sequence of the reporter plasmid expressing J3-gfp, J5-bfp, and J6-rfp is included in Supplementary Data 4. Source data are provided as a Source Data file.