Fig. 1: Designing inducible split Cas13 for orthogonal, multiplex, and composable RNA knockdown.

A The small molecule-inducible split Cas13 design comprises a small molecule-inducible dimerization/localization system, cellular compartment localization signals (NLS/NES), GS linkers, and split Cas13 ribonucleases. Reconstitution of Cas13 fragments is regulated by the small-molecule-induced dimerization/localization system in response to corresponding ligands. B Distinct features of the split Cas13 ribonuclease collection. The designed systems are tunable and achieve orthogonally regulated multiple transcript knockdown. When integrated with transcriptional control responsive to an endogenous pathway, the design system forms AND logic regulated by both endogenous and exogenous small molecules. When cascaded with other systems in the collection, they formed an incoherent feedforward loop (IFFL) where output expression adapts in response to a sustained small molecule signal. Finally, the designed system displayed robust orthogonally regulated multiplex knockdown in vivo. C The schematic shows the screening experimental setup. HEK293FT cells were transfected with plasmids encoding the GA-inducible split Cas13d systems, a targeting gRNA, mCh, and iRFP. Without the inducer, GA, transfected cells should express mCh strongly. When induced with GA (blue triangle), Cas13d splits should be reconstituted and become active to cleave mCh RNA resulting in weak mCh fluorescence. D Split sites were selected throughout the sequence of RfxCas13d in all domains. Ten out of 27 split sites screened with the GA-inducible dimerization domains generated inducibility >0.4 and suffered from leaky activity under no GA conditions. Data are presented as mean values +/− SEM. E Orientation of the GA-inducible dimerization domains was exchanged for GA-inducible split RfxCas13d designs with >0.4 inducibility. The orientation N Cas13d-GAI/GID-Cas13d C yielded higher inducibility with more split sites tested than the other orientation. And the design N507 Cas13d-GAI/GID-Cas13d 508 C had good performance with >0.8 inducibility and 0 leakiness. Data are presented as mean values +/− SEM. F Tagging N559 Cas13d with 2 NESs and 560 C Cas13d with 2 NLSs completely eliminated leaky activity generated with split N559/560 C in the original screening design while generating no negative impact on the inducibility. Tagging N559 Cas13d with an NES on the C-terminal achieved a similar performance, while using other combinations of NES and NLS led to reduced inducibility. The schematic shows the possible locations of NES or NLS for each design. 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.