Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

Elife. 2018 Dec 17:7:e41460. doi: 10.7554/eLife.41460.

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.

Keywords: Wnt signaling; cell biology; exosome; extracellular vesicle; genome wide screen; human; miRNA.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • A549 Cells
  • Base Sequence
  • CRISPR-Cas Systems
  • Endosomes / metabolism
  • Exocytosis
  • Exosomes / genetics
  • Extracellular Vesicles / genetics*
  • Gene Regulatory Networks
  • Genome, Human*
  • HEK293 Cells
  • HeLa Cells
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • MicroRNAs / genetics*
  • Multivesicular Bodies / metabolism

Substances

  • MicroRNAs