A microRNA-gated thgRNA platform for multiplexed activation of gene expression in mammalian cells

Abstract

Using a newly created AND-gate toehold-gated guide RNA (thgRNA), presence of specific miRNA and mRNA allows conditional gene activation and protein degradation.

Tunable and Modular miRNA Classifier through Indirect Associative Toehold Strand Displacement

“…This powerful strategy is generalizable to any strand displacement reaction and provides an initial framework for creating simple diagnostic assays. Beyond cell-free sensing applications, as the field makes progress in overcoming current intracellular delivery hurdles, associative strand displacement could serve as a strategy for creating miRNA-responsive devices that probe and control cellular behavior of live cells for in vivo applications . However, additional challenges beyond delivery include stability and half-life of the circuit in the cytosol, how to ensure all circuit components are delivered, and how the crowded intracellular environment affects the kinetics and output signal of the circuit.…”

“…Beyond cellfree sensing applications, as the field makes progress in overcoming current intracellular delivery hurdles, associative strand displacement could serve as a strategy for creating miRNA-responsive devices that probe and control cellular behavior of live cells for in vivo applications. 30 However, additional challenges beyond delivery include stability and halflife of the circuit in the cytosol, how to ensure all circuit components are delivered, and how the crowded intracellular ■ METHODS Materials. DNA oligos were purchased from Integrated DNA Technologies with standard desalting unless otherwise indicated.…”

Building RNA-Mediated Artificial Signaling Pathways between Endogenous Genes

“…Advances in CRISPR-based technologies have significantly broadened the spectrum of applicable RNA systems for construction of genetic circuits in both bacterial and mammalian cells. − Several papers have demonstrated the use of toehold-mediated strand displacement to activate Cas9 or Cas12 guide RNA (gRNA). − Generated by engineering different components of the guide RNA to create switchable RNA structures for controllable CRISPR functions, either exogenously introduced or endogenous RNA can be sensed as triggers to regulate the formation of active gRNA, typically through strand-displacement reactions. For example, toehold-gated gRNAs were proposed for activable CRISPR/Cas9 function in E. coli (Figure C). , A similar strategy is also applicable to the Cas12 system .…”

“…Recently, another circuit employing CRISPR/Cas12a was developed to avoid this limitation through the allosteric change of gRNA to achieve an “OFF-to-ON” switch of GFP signals in E. coli , in which the handle-blocking clamp acts as a trigger RNA senor, independently of the targeting region . Besides, CRISPR-mediated RNA circuits through strand binding and displacement have also been widely applied to sense RNA expression and regulate the target gene in mammalian cells and zebrafish embryos. ,,,− …”

Harnessing Catalytic RNA Circuits for Construction of Artificial Signaling Pathways in Mammalian Cells

“…However, how to employ RNA circuits to construct endogenous gene connections at the RNA level is rather difficult. Most previous studies on RNA-based genetic circuits rely on at least either an exogenous RNA X as the input or an engineered RNA Y as the target, [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] which are not suitable for endogenous gene connections. While exogenous RNAs can be engineered and controlled as desired, construction of a universal platform for manipulation of naturally occurring RNAs is a tough task.…”