Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

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Abstract

Extracellular vesicles mediate transfer of biologically active molecules between neighboring or distant cells, and these vesicles may play important roles in normal physiology and the pathogenesis of multiple disease states including cancer. However, the underlying molecular mechanisms of their biogenesis and release remain unknown. We designed artificially barcoded, exosomal microRNAs (bEXOmiRs) to monitor extracellular vesicle release quantitatively using deep sequencing. We then expressed distinct pairs of CRISPR guide RNAs and bEXOmiRs, enabling identification of genes influencing bEXOmiR secretion from Cas9-edited cells. This approach uncovered genes with unrecognized roles in multivesicular endosome exocytosis, including critical roles for Wnt signaling in extracellular vesicle release regulation. Coupling bEXOmiR reporter analysis with CRISPR-Cas9 screening provides a powerful and unbiased means to study extracellular vesicle biology and for the first time, to associate a nucleic acid tag with individual membrane vesicles.

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All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

Albert Lu

Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

For correspondence
alulopez@stanford.edu

Competing interests
No competing interests declared.
Paulina Wawro

Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

Competing interests
No competing interests declared.
David W Morgens

Department of Genetics, Stanford University School of Medicine, Stanford, United States

Competing interests
No competing interests declared.
Fernando Portela

Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0003-0238-9251
Michael C Bassik

Department of Genetics, Stanford University School of Medicine, Stanford, United States

Competing interests
No competing interests declared.
Suzanne R Pfeffer

Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

For correspondence
pfeffer@stanford.edu

Competing interests
Suzanne R Pfeffer, Reviewing editor, eLife.

"This ORCID iD identifies the author of this article:" 0000-0002-6462-984X

Funding

National Institutes of Health (NIDDK 37332)

Suzanne R Pfeffer

National Institutes of Health (DP2HD084069)

Michael C Bassik

National Institutes of Health (T32 HG000044)

David W Morgens

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.