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CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative
Karin Strohmeier1, Martina Hofmann1, Fabian Hauser1
1Department of Medical Engineering and Applied Social Sciences, University of Applied Sciences Upper Austria, Garnisonstraße 21, 4020 Linz, Austria.
International Journal of Molecular Sciences
|January 11, 2022
Summary
CRISPR/Cas9 genome editing offers precise fluorescent labeling of extracellular vesicles (EVs), overcoming limitations of traditional over-expression methods. This technique enables more accurate tracking and uptake studies of EVs, enhancing research reliability.
Area of Science:
- Biotechnology
- Cell Biology
- Nanotechnology
Background:
- Extracellular vesicles (EVs) are crucial for intercellular communication.
- Fluorescent labeling of EVs aids in studying their uptake and transport.
- Over-expression of fluorescent markers can lead to heterogeneous labeling and biased study results.
Purpose of the Study:
- To develop a precise method for fluorescently labeling extracellular vesicles (EVs).
- To overcome the limitations of heterogeneous labeling caused by over-expression techniques.
- To quantitatively compare CRISPR/Cas9-mediated labeling with conventional over-expression methods.
Main Methods:
- Utilized CRISPR/Cas9 genome editing to fluorescently label the EV marker CD63 with green fluorescent protein (GFP).
- Employed single-molecule sensitive fluorescence microscopy for quantitative analysis.
- Compared labeling efficiency and particle characteristics between CRISPR/Cas9-modified and over-expressing cells.
- Demonstrated flexibility by using HaloTag® and JaneliaFluor® 646 for 3D-localization microscopy.
Main Results:
- CRISPR/Cas9-mediated labeling resulted in a significantly higher fraction of single-GFP-labeled EVs (83%) compared to over-expression (36%).
- Single-labeled CRISPR-EVs were detectable and distinguishable from autofluorescence after cellular uptake.
- The HaloTag® labeling enabled advanced 3D-localization microscopy of single EV uptake.
Conclusions:
- CRISPR/Cas9 genome editing provides a superior, homogeneous approach for fluorescently labeling extracellular vesicles.
- This method minimizes artifacts in EV tracking and uptake studies.
- The developed technique offers flexibility for various labeling strategies and advanced imaging applications.
