Fig. 2: CRISPR protein expression optimisation and inducible CRISPRai toolkit architecture.

a + b, Impact of CRISPRai protein expression on gene activation and repression. a Low (small circle) and medium (large circle) strength promoter combinations used to drive the expression of dCas12a-VP (dark blue), dCas9-Mxi1 (blue), and Csy4 (light blue). The constitutive TDH3 promoter was used drive the transcription of an array containing an activation (dCas12a-VP) and repression (dCas9-Mxi1) gRNA targeting the RNR2 and TEF1 promoters driving the expression of mRuby2 and Venus, respectively. b Fluorescence measurements of all mid and low strength promoter-CRISPR protein combinations normalised to a control with no CRISPR proteins (No CRISPR). Experimental measurements are mRuby2 and Venus fluorescence levels per cell as determined by flow cytometry and shown as the mean ± SD from six biological replicates. c + d, Impact of CRISPRai protein expression on growth. c Low and medium strength promoter combinations used to drive the expression of the CRISPR proteins. d Maximum growth rates of all low and medium strength promoter combinations driving the expression of the CRISPR proteins and compared to a control with no CRISPR proteins (No CRISPR). Results are calculated maximum growth rates in YPD medium as determined from growth curves in a plate reader at OD₇₀₀ and are shown as the mean ± SD from four biological replicates. e Inducible CRISPRai vector architecture and gRNA array assembly. Inducible CRISPRai vector backbone (KanR-ColE1) not shown. f PCR generation of gRNA fragments for scarless BsaI Golden Gate assembly of gRNA arrays. Statistically significant differences between the all low strength promoter combination to all other promoter combinations were tested by one-way ANOVA, and significance levels are shown as p < 0.05 (*), p < 0.001 (***), p < 0.0001 (****).