Fig. 5: Cas13-based RNA-level IFFL pulse generator.

A The incoherent feed-forward loop, IFFL, is composed of an activation and a repression arm, where the activation arm, Dano-inhibited Cas13b, regulates both the output mCh and the repression arm Cas13d which also regulates output expression. In response to a sustained Dano-induction, output expression increases transiently and then undergoes adaptation, while the repression arm expression, GFP, consistently increases. B HEK293FT cells were transfected with the entire circuit, induced with Dano, and imaged for mCh and GFP mean fluorescent intensity (MFI) over time. As Cas13d-GFP expression consistently increased after Dano-induction, mCh adapted back to the basal expression level in response to the sustained Dano induction. Data are presented as mean values +/− SEM. Shaded area indicates the SEM for three biological replicates (n = 3). C Reducing Dano concentrations resulted in reduced adaptation responses of the circuit. Output expression normalized to mode is shown here, raw output expression data is shown in Supplementary Fig. 16C. Data are presented as mean values +/− SEM. Shaded area indicates the SEM for three biological replicates (n = 3). D A schematic showing different pulse topologies generated with varied small molecule concentrations regulating the activation arm. Parameters, such as the peak time, adaptation, and amplitude, can be used to quantitatively describe the pulse topology: higher concentrations of Dano, a stronger activation arm, may lead to faster peak time, increased peak amplitude, and decreased adaptation efficiency. E When we replace the repression arm of the IFFL with the GA-inducible Cas13d, the output pulse topology becomes susceptible to changes in both the activation regulator, Dano, and the repression arm regulator, GA. Heatmap shows the adaptation of output mCh at 60 h for different Dano and GA concentrations. With 5 uM Dano induction, output achieves perfect adaptation (~0%) at 60 h with minimal GA induction, as the repression arm is also expressed strongly along with the output. With a decreasing Dano concentration, a higher GA concentration is required for perfect adaptation. Experiment was repeated 3 times, and a representative experiment is displayed. F Peak time is affected by both Dano and GA in the dual-regulated IFFL. Output expression peaks sooner and starts to decrease in a short time with simultaneous strong induction of Dano and GA. Experiment was repeated 3 times, and a representative experiment is displayed. Source data are provided as a Source Data file.