RT Journal Article
SR Electronic
T1 Orthogonal inducible control of Cas13 circuits enables programmable RNA regulation in mammalian cells
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2023.03.20.533499
DO 10.1101/2023.03.20.533499
A1 Yage Ding
A1 Cristina Tous
A1 Jaehoon Choi
A1 Jingyao Chen
A1 Wilson W. Wong
YR 2023
UL http://biorxiv.org/content/early/2023/03/20/2023.03.20.533499.abstract
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.