Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping

Nat Commun. 2020 Mar 13;11(1):1334. doi: 10.1038/s41467-020-14957-y.


Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Cas Systems*
  • Cell Survival
  • Dimerization
  • Exons / genetics*
  • Extracellular Vesicles / metabolism*
  • Gene Editing
  • Genetic Vectors / metabolism
  • HEK293 Cells
  • HIV Protease / metabolism
  • Humans
  • Induced Pluripotent Stem Cells / metabolism
  • Ligands
  • Luciferases / metabolism
  • Nanoparticles / chemistry*
  • RNA Splicing / genetics
  • RNA, Catalytic / metabolism
  • RNA, Guide, Kinetoplastida / metabolism*
  • Ribonucleoproteins / metabolism
  • Tissue Donors
  • tat Gene Products, Human Immunodeficiency Virus / metabolism


  • Ligands
  • RNA, Catalytic
  • RNA, Guide
  • Ribonucleoproteins
  • tat Gene Products, Human Immunodeficiency Virus
  • Luciferases
  • CRISPR-Associated Protein 9
  • HIV Protease