Fig. 6. Control of a CRISPRi inverter with competitor binding sites.
A Top: Diagram showing how production of a dCas12a CRISPR RNA (crRNA) is controlled by an aTc-inducible promoter, and the dCas12a+crRNA duplex can either bind to the active site Sa, which turns off production of sfGFP, or to a decoy site Sd residing on a “sponge” plasmid. Bottom: Fluorescence intensity vs time for CRISPR-dCas12a systems with variable numbers of sponge sites (Nc). B Induction curves for a CRISPRi system with sponge sites on plasmid with varying copy numbers, n = 3 biological replicates for each data point. C ON-OFF levels for variable numbers of sponge sites. The ON and OFF fluorescence level of the inverter both increase as the number of sponge sites varies from 0 to 270. The dynamic range decreases with increasing sponge copy number. D Active site occupancy vs number of sponge sites, as the number of sponge sites increases the active site becomes less occupied. The gray area represents the numerical solution of a fold-change model15,35 for dCas12a where the binding energy equal to − 13.25 kBT (top) and − 12.25 kBT (bottom).