Orthogonal inducible control of Cas13 circuits enables programmable RNA regulation in mammalian cells

Introduction:: RNA plays an indispensable role in mammalian cell functions. Cas13, a class of RNA-guided ribonuclease, is a flexible tool for modifying and regulating coding and non-coding RNAs, with enormous potential for creating new cell functions. However, the lack of control over Cas13 activity has limited its cell engineering capability. Here, we present the CRISTAL (Control of RNA with Inducible SpliT CAs13 Orthologs and Exogenous Ligands) platform. CRISTAL is powered by a collection (10 total) of orthogonal split inducible Cas13s that can be turned ON or OFF via small molecules in multiple cell types, providing precise temporal control. Also, we engineered Cas13 logic circuits that can respond to endogenous signaling and exogenous small molecule inputs. Furthermore, the orthogonality, low leakiness, and high dynamic range of our inducible Cas13d and Cas13b enable the design and construction of a robust incoherent feedforward loop, leading to near-perfect and tunable adaptation response. Finally, using our inducible Cas13s, we achieve simultaneous multiplexed control of multiple genes in vitro and in mice. Together, our CRISTAL design represents a powerful platform for precisely regulating RNA dynamics to advance cell engineering and elucidate RNA biology.

Materials and Methods:: We predicted the structure of the Cas13d derived from Ruminococcus flavefaciens XPD3002 (RfxCas13d) based on that of the Cas13d from Eubacterium siraeum (EsCas13d). Based on the prediction, we selected 27 split sites to avoid secondary structures and conserved regions (Figure 1a). We used the gibberellic acid (GA)-inducible dimerization domains (GID and GAI) to screen for the inducible activity of the split RfxCas13d (Figure 2a). To test the activity of the designs, we transfected HEK293FT cells with plasmids encoding the GA-inducible split Cas13d pairs, a mCherry-targeting gRNA, mCh, and iRFP as a transfection marker. We measured the mCh and iRFP mean fluorescent intensity via flow cytometry (Figure 2b). Two attributes of a potent inducible system are strong induction and low leakiness. We defined system leakiness as the difference between 1, and the relative mCherry MFI of the uninduced state, representing mCherry-specific knockdown caused by the split Cas13d in the absence of GA (Figure 2c). Inducibility is defined as the difference between the relative mCh MFI of an induced and uninduced condition, normalized to the uninduced condition (Figure 2c). Therefore, an ideal inducible system should have leakiness = 0 and inducibility = 1 (Figure 2d). Designs that yield larger than 0.4 inducibility were further optimized through CID orientation exchange and cellular compartment localization for minimal leakiness and improving inducibility.

Results, Conclusions, and Discussions:: Using several chemical inducible dimerization (CID) systems and Cas13 orthologs, we created 10 different chemical-inducible systems that offer efficient knockdown at the protein level up to 78% in vitro and up to 98% in mice against an abundantly expressed transgene using non-toxic plant hormones or FDA-approved drugs. Of the designed systems, we characterized the tunable and reversible activity and the ability to multiplex endogenous target knockdown (Figure 1). In the future, we anticipate that using the growing family of Cas13 systems as the RNA regulatory module and orthogonal CID systems as the exogenous control module will facilitate the expansion of our toolkit, more sophisticated cellular reprogramming, and clinical translation of such processes.

To demonstrate the robust activity of our toolkit in more sophisticated circuits, we applied our systems for AND logic that only cleaves RNA transcripts in the presence of both an exogenous chemical and an endogenous pathway (WNT)-activating signal (Figure 1b, 3). We also applied our systems for multiplex and demultiplex regulation and implemented an incoherent feed-forward loop utilizing two orthogonal inducible Cas13s to create a near-perfect and tunable adaptation response (Figure 1b, 4). As the output can be easily switched from a transgene to an endogenous gene, our system can render the endogenous transcriptome under complex regulation.

Finally, we achieved orthogonal regulated multiplex RNA knockdown in mice through transient expression of the inducible systems (Figure 1, 5). The robust activity of our systems in vivo for orthogonal and multiplexed RNA regulation unleashes the potential for constructing complex circuits in vivo. In the future, stable integration methods such as the PiggyBac transposon system may allow us to generate animal models with sustained system expression and activity.

Together, our CRISTAL platform has the potential to facilitate complex circuit engineering in vivo with custom-designed transcriptome profiles for basic studies and therapeutic applications.


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