CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative Analysis

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 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.

EVAnalyzer: High content imaging for rigorous characterisation of single extracellular vesicles using standard laboratory equipment and a new open‐source ImageJ/Fiji plugin

“…We used EVs from HEK293T cells engineered to express different versions of FP‐tagged CD63. This included stable, random integration of CD63‐mNeon (Wiklander et al., 2019 ) or CD63‐GFP (‘OE’, (Strohmeier et al., 2021 )), as well as transient transfection of CD63‐emGFP. In addition, we used a HEK293T line with GFP engineered into the CD63 locus by CRISPR/Cas9 technology (‘CD63‐GFP CRISPR’) (Strohmeier et al., 2021 ).…”

“…This included stable, random integration of CD63‐mNeon (Wiklander et al., 2019 ) or CD63‐GFP (‘OE’, (Strohmeier et al., 2021 )), as well as transient transfection of CD63‐emGFP. In addition, we used a HEK293T line with GFP engineered into the CD63 locus by CRISPR/Cas9 technology (‘CD63‐GFP CRISPR’) (Strohmeier et al., 2021 ). EVs from these different parent cells were isolated by UF‐SEC (Supplementary File 3 ) and analysed by Fluorescence Correlation Spectroscopy (FCS) to determine the average number of fluorescent molecules per vesicle, following a previously described protocol (Corso et al., 2019 ).…”

“…Single vesicles can be detected and quantified as light diffraction limited spots after intrinsic labelling with FP‐tagged marker proteins (Corso et al., 2019 ; Heusermann et al., 2016 ) or immunostaining (Bağcı et al., 2022 ; Burbidge et al., 2020 ; Han et al., 2021 ; Hartjes et al., 2020 ) when immobilised on optical surfaces. More recent studies have focused on more advanced microscopy techniques for imaging of individual EVs using custom developed TIRF (Han et al., 2021 ) or single molecule localisation microscopy setups (Strohmeier et al., 2021 ). Despite the development of these highly specialised applications, the fact that even standard widefield fluorescence microscopes can be used (Corso et al., 2019 ) provided an ideal starting point to derive a more generically usable, straightforward process for single vesicle quantification with standard laboratory equipment.…”

“…For all transfection experiments performed throughout the manuscript, the ratio DNA: PEI is kept constant (1 μg DNA: 1.5 μg PEI). Additionally, Evs were isolated from HEK293T cells stably expressing CD63-mNeon kindly provided by Dr. Samir El Andaloussi and Dr. Andre Görgens (Karolinska Institute, Stockholm, Sweden;(Corso et al, 2017;Dar et al, 2021), as well as from HEK293T cells with CRISPR-Cas9 mediated genomic integration of GFP into the CD63 locus ('CD63 CRISPR') or random genomic integration of CD63-GFP kindly provided by Dr. Mario Maierhofer (Fachhochschule Upper Austria, Linz, Austria; (Strohmeier et al, 2021)). Cells were cultured in DMEM supplemented with 10% FBS, 1% GlutaMAX and 1% Antibiotic/Antimycotic.…”

Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy

“…The uptake of these EVs was previously confirmed in HeLa cells; they remain mainly in the cytosol and in proximity to the nucleus . Nevertheless, no information on the uptake dynamics of EVs is yet available.…”