PT  - JOURNAL ARTICLE
AU  - Yage Ding
AU  - Cristina Tous
AU  - Jaehoon Choi
AU  - Jingyao Chen
AU  - Wilson W. Wong
TI  - Orthogonal inducible control of Cas13 circuits enables programmable RNA regulation in mammalian cells
AID  - 10.1101/2023.03.20.533499
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2023.03.20.533499
4099  - http://biorxiv.org/content/early/2023/03/20/2023.03.20.533499.short
4100  - http://biorxiv.org/content/early/2023/03/20/2023.03.20.533499.full
AB  - 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.Competing Interest StatementA patent application has been filed based on this work (Y.D and W.W.W). W.W.W. is a scientific co-founder and shareholder of Senti Biosciences and 4Immune.