Department of Medical Engineering and Applied Social Sciences, University of Applied Sciences Upper Austria, Garnisonstraße 21, 4020 Linz, Austria.
Center of Excellence Food Technology and Nutrition, University of Applied Sciences Upper Austria, Stelzhamerstraße 23, 4600 Wels, Austria.
Int J Mol Sci. 2021 Dec 28;23(1):282. doi: 10.3390/ijms23010282.
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.
荧光标记物的过度表达常用于可视化囊泡的摄取和运输。通过过度表达标记的 EV 在单个颗粒上的荧光团数量方面表现出相当大的异质性，这可能会偏向于跟踪和摄取研究，有利于标记更强烈的颗粒。为了避免过度表达引起的潜在伪影，我们使用 CRISPR/Cas9 技术开发了一种用于用绿色荧光蛋白对细胞外囊泡标记物 CD63 进行荧光标记的基因组编辑方法。使用单分子敏感荧光显微镜，我们使用真正的单颗粒测量对常规过度表达和 CRISPR/Cas9 方法分泌的小细胞外囊泡的标记程度进行了定量比较。通过我们的分析，可以证明与从 GFP-CD63 过表达细胞中分离的 EV（36%）相比，从 CRISPR/Cas9 修饰的细胞中分离的 EV 中具有更大比例的单 GFP 标记 EV（83%）。尽管只有单 GFP 标记，但 CRISPR-EV 在被细胞摄取后可以从自体荧光中被检测和区分。为了证明 CRISPR/Cas9 基因组编辑方法的灵活性，我们使用脂质膜可渗透染料 JaneliaFluor 646 对 EV 进行了 HaloTag 荧光标记，这使我们能够对培养细胞摄取的单个 EV 进行 3D 定位显微镜检查。
