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Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells
Published on: July 6, 2021
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Optogenetic Control of Background Fluorescence Reduction for CRISPR-Based Genome Imaging
Yu Hou1,2, Dianbing Wang1, Song Lu1
1National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Analytical Chemistry
|June 6, 2022
Summary
Researchers developed a new CRISPR-based method to improve live-cell imaging of DNA. This optogenetic strategy reduces background noise, enabling clearer visualization of low-copy genomic loci.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- CRISPR/dCas9 is vital for live-cell genomic imaging.
- High nuclear background noise limits imaging of low-copy or non-repetitive DNA loci.
Purpose of the Study:
- To develop an optogenetically controlled method to reduce background fluorescence for improved genomic locus imaging.
- To enhance the signal-to-noise ratio for visualizing challenging genomic targets.
Main Methods:
- Combined CRISPR-SunTag system with a light-inducible nuclear export tag (LEXY).
- Recruited LEXY-tagged sfGFP to dCas9 targeting specific genomic loci.
- Used blue light to trigger nuclear export of untargeted fluorescent modules to the cytoplasm.
Main Results:
- Successfully visualized genomic loci with as few as nine repeat copies.
- Achieved a significant increase in the signal-to-noise ratio.
- Demonstrated light-controllable reduction of nuclear background fluorescence.
Conclusions:
- The proposed optogenetic strategy effectively reduces background noise in live-cell imaging.
- This method enhances the visualization of low-copy genomic loci.
- The technique offers a simple, controllable approach with broad applications in cell biology.
