A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA

Abstract

Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small noncoding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.

Growing pains: addressing the pitfalls of plant extracellular vesicle research

“…They cannot be removed sufficiently by differential centrifugation (Auber et al ., 2019), and while high‐resolution density gradients can help separate out some nonvesicle particles (Jeppesen et al ., 2019), other particles, such as miRNA‐associated lipoprotein complexes, have been found to co‐purify with vesicle‐rich fractions (Sodar et al ., 2016). Because many extracellular RNAs are associated with protein complexes, RNase treatment is usually only effective following a proteinase treatment (Arroyo et al ., 2011; Shurtleff et al ., 2017); thus, RNase protection assays should be performed after protease treatment (de Jong et al ., 2020). Validating EV RNA cargo requires multiple controls and considerations, as detailed in Box 3.…”

Emerging Gene-Editing Modalities for Osteoarthritis

“…Genome-editing factors, packaged in engineered CRISPR/Cas9 complexes, can be enclosed in extracellular vesicles (EVs), for delivery to specific target cells [ 86 , 87 ]. EVs are composed of cellular constituents such as lipids, proteins, RNA, and DNA [ 86 , 88 ].…”

“…EVs are composed of cellular constituents such as lipids, proteins, RNA, and DNA [ 86 , 88 ]. EVs may cross the blood–brain barrier, target cells in vivo, and protect their components from degradation in the circulatory system [ 87 , 89 ]. Their function is dependent upon their origin, and EVs derived from MSCs could have the potential to deliver contents to OA cells [ 90 , 91 , 92 , 93 ], as shown in Figure 3 [ 85 , 94 , 95 ].…”

Mastering the Tools: Natural versus Artificial Vesicles in Nanomedicine

“…[13] Interestingly, the relevance of EVs is witnessed by their conservation during evolution from bacteria to superior eukaryotes, such as human and plants, and their ability to mediate even the "inter-kingdom" communication. [24] Several biomolecules may be carried within the lumen of EVs, such as DNA (both mitochondrial and genomic), [25,26] RNA (including mRNA, miRNA, siRNA, and lncRNA), [27][28][29][30] lipids and functional proteins (such as active enzymes). [23] These cargoes are protected from degradation by proteases and nucleases, commonly present in the extracellular environment, allowing for an efficient delivery of active biomolecules to target cells.…”