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Updated: Jun 11, 2025
Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells
Published on: July 6, 2021
High-resolution and programmable RNA-IN and RNA-OUT genetic circuit in living mammalian cells
Min Zhang1, Xue Zhang1, Yongyue Xu1
1MOE Key Laboratory of Bioinformatics, Center for Synthetic and System Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Abstract:
RNAs and their encoded proteins intricately regulate diverse cell types and states within the human body. Dysregulated RNA expressions or mutations can lead to various diseased cell states, including tumorigenesis. Detecting and manipulating these endogenous RNAs offers significant promise for restoring healthy cell states and targeting tumors both in research and clinical contexts. This study presents an RNA-IN and RNA-OUT genetic circuit capable dynamically sensing and manipulating any RNA target in a programmable manner. The RNA-IN module employes a programmable CRISPR-associated protease (CASP) complex for RNA detection, while the RNA-OUT module utilizes an engineered protease-responsive dCas9-VPR activator. Additionally, the CASP module can detect point mutations by harnessing an uncovered dual-nucleotide synergistic switching effect within the CASP complex, resulting in the amplification of point-mutation signals from initially undetectable levels (1.5-fold) to a remarkable 94-fold. We successfully showcase the circuit's ability to rewire endogenous RNA-IN signals to activate endogenous progesterone biosynthesis pathway, dynamically monitor adipogenic differentiation of mesenchymal stem cells (MSCs) and the epithelial-to-mesenchmal trans-differentiation, as well as selective killing of tumor cells. The programmable RNA-IN and RNA-OUT circuit exhibits tremendous potential for applications in gene therapy, biosensing and design of synthetic regulatory networks.
Insights
This study introduces a programmable RNA-IN and RNA-OUT genetic circuit for sensing and manipulating cellular RNAs. This innovation enables precise detection of mutations and dynamic control of cellular processes, offering new therapeutic avenues.
Area of Science:
- Synthetic Biology
- Molecular Biology
- Gene Regulation
Background:
- Cellular functions are regulated by RNA and protein expression.
- Aberrant RNA expression and mutations contribute to diseases like cancer.
- Targeting endogenous RNAs holds therapeutic potential for disease treatment.
Purpose of the Study:
- To develop a programmable genetic circuit for dynamic RNA sensing and manipulation.
- To enhance the detection sensitivity of point mutations.
- To demonstrate the circuit's utility in controlling cellular pathways and states.
Main Methods:
- Development of an RNA-IN module using a programmable CRISPR-associated protease (CASP) complex for RNA detection.
- Implementation of an RNA-OUT module with an engineered protease-responsive dCas9-VPR activator.
- Harnessing a dual-nucleotide synergistic switching effect within the CASP complex for mutation signal amplification.
Main Results:
- The CASP module amplified point-mutation signals by up to 94-fold.
- The RNA-IN/RNA-OUT circuit successfully rewired endogenous RNA signals to control progesterone biosynthesis.
- Demonstrated dynamic monitoring of mesenchymal stem cell (MSC) differentiation and epithelial-to-mesenchymal transition (EMT), alongside selective tumor cell killing.
Conclusions:
- The developed programmable RNA-IN and RNA-OUT circuit offers dynamic control over cellular RNA.
- This system significantly enhances point mutation detection sensitivity.
- The circuit shows broad potential for gene therapy, biosensing, and synthetic regulatory network design.
