Fig. 1

NOT gates used to test the action of dSpCas9(-Mxi1):gRNA on the synthetic promoter Tsynth8_pCYC1noTATA. a Circuit scheme. The guide RNA is expressed either via an RGR cassette or an RNA polymerase III-dependent promoter (pSNR52 or pRPR1). Galactose induces the synthesis of dSpCas9(-Mxi1). b Ratio between the NOT gate fluorescence level in the presence (OFF state) and absence (ON state) of galactose. Each gate is labelled with the expression system for the guide RNA (RGR cassette–red color; pSNR52i: pSNR52-gRNA-SUP4t on an integrative plasmid–blue; pSNR52m: pSNR52-gRNA-SUP4t on a multicopy plasmid–green; pRPR1i: pRPR1-gRNA-RPR1t on an integrative plasmid–orange; pRPR1m: pRPR1-gRNA-RPR1t on a multicopy plasmid–orange) followed by Mxi1 when this repression domain was attached to dSpCas9. The highest fluorescence repression was obtained by expressing the guide RNA via the SNR52 promoter on an integrative plasmid together with dSpCas9 fused to Mxi1 (pSNR52i-Mxi1). This NOT gate configuration clearly outperforms the other seven (the “ ∗∗” symbol on top of the corresponding bar indicates a statistically significant difference from all the other constructs–two-sided Welch’s t-test, p-value < 0.05). Each relative fluorescence level is the mean value obtained from 3 up to 6 independent experiments (i.e. carried out in different days). Further statistical considerations are reported in Additional file 1: Table S1 and Figure S1. c Normalized gRNA expression from the three NOT gate schemes selected to build the anti-CRISPR-based biosensors. For each gate, the relative amount of gRNA with respect to the mRNA produced by the ACT1 gene was first calculated (mean value from three replicates during a single experiment– 12.5 ng of cDNA were used). Then, gRNA relative expressions were normalized to the value obtained for pSNR52i-Mxi1. Error bars were determined on the normalized values via the error propagation formula. The ADH1 promoter present in the RGR-Mxi1 configuration appears to drive the synthesis of as much gRNA (1.02 ±0.26) as the RNA polymerase III-dependent SNR52 promoter on an integrative plasmid i.e. the reference strain pSNR52i-Mxi1 to which the value 1.00 (± 0.26) is assigned (two-sided Welch’s t-test, p-value =0.96). In contrast, pSNR52m-Mxi1–where pSNR52 is placed on a multicopy plasmid–expresses 12.99 (± 3.15)-fold more gRNA than pSNR52i-Mxi1 (the “ ∘” symbol points out a statistically significant difference with respect to the reference gate–two-sided Welch’s t-test, p-value < 0.05). Thus, gRNA expression is not directly correlated with NOT gate efficiency since lower gRNA levels–as in pSNR52i-Mxi1 and RGR-Mxi1–correspond to higher fluorescence repression