Fig. 4: Orthogonal multiplex inducible RNA regulation.

A Collection of optimized small molecule-inducible split Cas13b systems responsive to GA, ABA, and Dano. All ON and OFF switches with Cas13b generate mCh-specific knockdown with a dynamic range of around 70% between induced and uninduced conditions and no leaky activity in the OFF state. B Dano-inducible Cas13b regulates mCh expression, while GA-inducible Cas13d orthogonally regulates HLA surface expression through targeted knockdown of B2M. Results show ~60% reduced mCh expression only upon Dano induction, while HLA expression is downregulated by >80% with GA induction. No cross-talk between the GA-inducible Cas13d and Dano-inducible Cas13b was observed. Dual knockdown of mCh and HLA upon simultaneous stimulation of GA and Dano is as efficient as the knockdown when induced separately. C Dano-inducible Cas13b regulates mCh expression while Dano-inhibited Cas13d regulates HLA surface knockdown. Upon Dano induction, Cas13b is actively knocking down mCh, while Cas13d is inactive. In the absence of Dano, Cas13d inhibits HLA expression while Cas13b is inactive, and mCh is highly expressed. 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.