L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma.

L1CAM is a transmembrane protein expressed on neurons that was presumed to be found on neuron-derived extracellular vesicles (NDEVs) in human biofluids. We developed a panel of single-molecule array assays to evaluate the use of L1CAM for NDEV isolation. We demonstrate that L1CAM is not associated with extracellular vesicles in human plasma or cerebrospinal fluid and therefore recommend against its use as a marker in NDEV isolation protocols.

Comparison of Cas9 activators in multiple species.

Several programmable transcription factors exist based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems, and we assess the role of cooperativity in maximizing gene expression.

Cas9 gRNA engineering for genome editing, activation and repression.

We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

Extracellular Vesicle Isolation and Analysis by Western Blotting.

Extracellular vesicles (EVs) are released by mammalian cells and are thought to be important mediators of intercellular communication. There are many methods for isolating EVs from cell culture media, but the most popular methods involve purification based on ultracentrifugation . Here, we provide a detailed protocol for isolating EVs by differential ultracentrifugation and analyzing EV proteins (such as the tetraspanins CD9 , CD63 and CD81 ) by western blotting.

Imaging of Isolated Extracellular Vesicles Using Fluorescence Microscopy.

High-resolution fluorescence microscopy approaches enable the study of single objects or biological complexes. Single object studies have the general advantage of uncovering heterogeneity that may be hidden during the ensemble averaging which is common in any bulk conventional biochemical analysis. The implementation of single object analysis in the study of extracellular vesicles (EVs) may therefore be used to characterize specific properties of vesicle subsets which would be otherwise undetectable. We present a protocol for staining isolated EVs with a fluorescent lipid dye and attaching them onto a glass slide in preparation for imaging with total internal reflection fluorescence microscopy (TIRF-M) or other high-resolution microscopy techniques.

Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.

The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge - of the nature of EV(-RNA)s and of how to effectively and reliably study them - currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data.