Fig. 3: Multiplex control of split Cas13d ribonucleases.
A The top panel illustrates 4 optimized split RfxCas13d designs responsive to Dano, 4-OHT, and ABA. The dNS3/DNCR domains dimerize upon Dano induction, resulting in ~60% mCh-specific knockdown. The ERT2 domain localizes to the nucleus upon 4OHT induction, bringing the Cas13d C-terminal in close proximity with the N-terminal that is sequestered in the nucleus with an NLS, leading to ~60% induced mCh-specific knockdown with minimal leaky activity when uninduced. The ABI/PYL system dimerizes upon ABA induction, leading to 67% mCh-specific knockdown. While the former 3 systems are ON switches that become active upon induction, the dNS3/ANR system forms an OFF switch with split Cas13d. The dNS3/ANR system constitutively dimerizes until Dano competitively binds to dNS3 and displaces ANR, which inhibits Cas13d activity. Constitutive mCh-specific knockdown was over 70%, which is fully rescued with Dano induction. B Schematic of the small molecule-endogenous signal AND gate. A GSK inhibitor (green circles), mimicking the endogenous WNT signaling pathway, activates the WNT-responsive SuperTOP Flash (STF) promoter and activates the transcription of the GA-inducible split Cas13d. This Cascading transcriptional and post-translational control of split Cas13d achieved ~70% mCh-specific knockdown only in the presence of both the GSK inhibitor and GA (blue triangle). Data are presented as mean values +/− SEM. Error bars indicate the SEM for three biological replicates (n = 3). Source data are provided as a Source Data file.
