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Small-Molecule-Mediated Split-Aptamer Assembly for Inducible CRISPR-dCas9 Transcription Activation
Xiao-Han Liu1, Bang-Rui Li1, Zhan-Ming Ying1
1State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
ACS Chemical Biology
|June 14, 2022
Summary
We developed a novel CRISPR-dCas9 system that uses split aptamers to detect S-adenosyl methionine (SAM) in live cells. This inducible system enables quantitative imaging and regulation of gene expression based on SAM levels.
Area of Science:
- Molecular Biology
- Biotechnology
- Gene Regulation
Background:
- CRISPR-dCas9 systems are versatile tools for transcription regulation and sensing.
- Developing inducible systems for metabolite detection in live cells remains a challenge.
Purpose of the Study:
- To engineer a small-molecule-mediated split-aptamer assembly for inducible CRISPR-dCas9 transcription activation.
- To enable quantitative detection and live-cell imaging of S-adenosyl methionine (SAM).
Main Methods:
- Integrated split SAM aptamer fragments into guide RNA (gRNA) and MS2 arrays.
- Utilized SAM-mediated reassembly to recruit an MCP-fused transcription activator to the dCas9 complex.
- Quantified SAM levels and monitored gene expression via a near-infrared fluorescent protein.
Main Results:
- Demonstrated quantitative detection of SAM in live cells with high sensitivity.
- Confirmed that MAT1A and MAT2A catalyze SAM production in live cells.
- Showed that SAM levels in cancer cells can be increased by upregulating MAT1A mRNA.
Conclusions:
- The split-aptamer assembly strategy offers a new approach for controlling CRISPR-dCas9 systems.
- This method enables conditional transcription regulation in response to endogenous metabolites.
- The system provides a powerful tool for studying SAM metabolism and its role in cellular processes.
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