Crispr/cas9 genome editing vs. Over-expression for fluorescent extracellular vesicle-labeling: A quantitative analysis

Karin Strohmeier, Martina Hofmann, Fabian Hauser, Dmitry Sivun, Sujitha Puthukodan, Andreas Karner, Georg Sandner, Pol Edern Le Renard, Jaroslaw Jacak, Mario Mairhofer

Research output: Contribution to journalArticlepeer-review

Abstract

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 conven-tional 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.

Original languageEnglish
Article number282
JournalInternational Journal of Molecular Sciences
Volume23
Issue number1
DOIs
Publication statusPublished - 28 Dec 2021

Keywords

  • Atomic force microscopy
  • CD63
  • CRISPR/Cas9
  • Extracellular vesicles
  • Genome editing
  • Single-molecule fluorescence microscopy
  • Single-molecule labeling stoichiometry
  • Humans
  • Fluorescence
  • Extracellular Vesicles/metabolism
  • Gene Editing
  • Green Fluorescent Proteins/metabolism
  • CRISPR-Cas Systems/genetics
  • Staining and Labeling
  • HEK293 Cells
  • HeLa Cells

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