Multimodal control of Cas13d activity through domain insertion at an allosteric hotspot

CRISPR-Cas13d RNA nucleases are powerful tools for programmable RNA targeting. A light-controlled RNA nuclease could be transformative by enabling researchers to selectively knock down transcripts at desired positions in a cell or tissue or at timepoints of interest. Here, we develop a set of RfxCas13d tools that can be multimodally controlled by either light or small molecule addition. By screening an RfxCas13d library containing insertions of the AsLOV2 photoswitchable domain, we identify an OptoCas13d-off variant that induced target RNA cleavage in the dark and switched to an inactive state under blue light. We show that the same allosteric hotspot can be exploited to generate an OptoCas13d-on with an inverted light response and a ChemoCas13d that is activated by rapamycin analogs, enabling knockdown of endogenous mRNA and protein targets. Overall, our study shows that engineered allostery can produce stimulus-controlled Cas13d variants to modulate RNA with high spatial and temporal precision.