CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative Analysis
| Autor: | Karin Strohmeier, Martina Hofmann, Fabian Hauser, Dmitry Sivun, Sujitha Puthukodan, Andreas Karner, Georg Sandner, Pol-Edern Le Renard, Jaroslaw Jacak, Mario Mairhofer |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Gene Editing
atomic force microscopy Staining and Labeling QH301-705.5 single-molecule fluorescence microscopy Green Fluorescent Proteins Article Fluorescence Chemistry Extracellular Vesicles HEK293 Cells single-molecule labeling stoichiometry CD63 genome editing Humans Biology (General) CRISPR-Cas Systems QD1-999 CRISPR/Cas9 HeLa Cells |
| Zdroj: | International Journal of Molecular Sciences International Journal of Molecular Sciences, Vol 23, Iss 282, p 282 (2022) |
| ISSN: | 1422-0067 |
| Popis: | Over-expression of fluorescently-labeled markers for extracellular vesicles is frequently used to visualize vesicle up-take and transport. EVs that are labeled by over-expression show considerable heterogeneity regarding the number of fluorophores on single particles, which could potentially bias tracking and up-take studies in favor of more strongly-labeled particles. To avoid the potential artefacts that are caused by over-expression, we developed a genome editing approach for the fluorescent labeling of the extracellular vesicle marker CD63 with green fluorescent protein using the CRISPR/Cas9 technology. Using single-molecule sensitive fluorescence microscopy, we quantitatively compared the degree of labeling of secreted small extracellular vesicles from conventional over-expression and the CRISPR/Cas9 approach with true single-particle measurements. With our analysis, we can demonstrate a larger fraction of single-GFP-labeled EVs in the EVs that were isolated from CRISPR/Cas9-modified cells (83%) compared to EVs that were isolated from GFP-CD63 over-expressing cells (36%). Despite only single-GFP-labeling, CRISPR-EVs can be detected and discriminated from auto-fluorescence after their up-take into cells. To demonstrate the flexibility of the CRISPR/Cas9 genome editing method, we fluorescently labeled EVs using the HaloTag® with lipid membrane permeable dye, JaneliaFluor® 646, which allowed us to perform 3D-localization microscopy of single EVs taken up by the cultured cells. |
| Databáze: | OpenAIRE |
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