Abstract
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.
Highlights
Prolonged expression of the Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas[9] nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity
Lyn mystoylation (LM)-FKBP12-EGFP was selected as a candidate because of its ability to target the plasma membrane of cells and potential to be passively packaged into budding extracellular vesicles (EVs)
The second utilizes an HIV Ψ packaging signal to direct sgRNA flanked by HH and hepatitis delta virus (HDV) selfcleaving ribozymes into NanoMEDIC through an interaction with
Summary
Prolonged expression of the CRISPR-Cas[9] nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. CRISPR-Cas[9] has been reported as an efficient tool for inducing exon skipping in iPSCs5 and in vivo animal DMD models[6,7,8,9] to restore dystrophin protein expression. Adeno-associated viruses (AAV) have been the leading tool for in vivo gene delivery, and utilized to treat DMD animal models by delivering the CRISPR-Cas[9] system[6,7,8,9]. Ribonucleoprotein (RNP) delivery of CRISPR-Cas[9] offers several advantages over DNA delivery[19] It facilitates potent ontarget cleavage while reducing unwanted off-target effects, as RNP is rapidly degraded in cultured cells compared with DNA plasmid expression vectors[20]. There is a need for active incorporation machinery for Cas[9] protein and sgRNA, which does not involve direct fusion of Cas[9] protein with Gag
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