Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities

Abstract

In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome editing systems have become one of the most robust platforms in basic biomedical research and therapeutic applications. To date, efficient in vivo delivery of the CRISPR/Cas9 system to the targeted cells remains a challenge. Although viral vectors have been widely used in the delivery of the CRISPR/Cas9 system in vitro and in vivo, their fundamental shortcomings, such as the risk of carcinogenesis, limited insertion size, immune responses and difficulty in large-scale production, severely limit their further applications. Alternative non-viral delivery systems for CRISPR/Cas9 are urgently needed. With the rapid development of non-viral vectors, lipid- or polymer-based nanocarriers have shown great potential for CRISPR/Cas9 delivery. In this review, we analyze the pros and cons of delivering CRISPR/Cas9 systems in the form of plasmid, mRNA, or protein and then discuss the limitations and challenges of CRISPR/Cas9-based genome editing. Furthermore, current non-viral vectors that have been applied for CRISPR/Cas9 delivery in vitro and in vivo are outlined in details. Finally, critical obstacles for non-viral delivery of CRISPR/Cas9 system are highlighted and promising strategies to overcome these barriers are proposed.

Keywords: CRISPR/Cas9; Cancer; Clinical translation; Genetic disorder; Nanomedicine; Non-viral delivery.

Fig. 1

Schematic illustration of the two different repair mechanisms of CRISPR/Cas9-mediated double stranded breaks (DSBs).

Fig. 2

Non-viral vectors for in vitro CRISPR/Cas9 delivery. (A) Rational design of arginine nanoparticles (ArgNPs) for intracellular delivery of engineering Cas9 protein (Cas9En) or Cas9En/sgRNA ribonucleoprotein complexes (RNPs) via membrane fusion. Engineering Cas9 protein was constructed to carry an N-terminus E-tag and a C-terminus nuclear localization signal (NLS). Reprinted with permission from ref. . Copyright 2017 American Chemical Society. (B) The schematic process of preparation, cellular uptake, endosomal eacape and genome editing of zeolitic imidazole frameworks-based CRISPR/Cas9 (CC-ZIFs) delivery. Reprinted with permission from ref. . Copyright 2017 American Chemical Society. (C) Design of modular RNA aptamer-streptavidin complexes (S1mplexes) for co-delivery of Cas9/sgRNA ribonucleoprotein complexes and ssODN donor template. Reprinted with permission from ref. . Copyright 2015 Nature Publishing Group. (D) The schematic process of preparation and intracellular delivery of GO-PEG-PEI based Cas9/sgRNA delivery. The confocal laser scanning images and the quantification of fluorescence intensity indicated down-regulated expression of EGFP protein after targeted knockout of EGFP genes in AGS.EGFP cells with GO-PEG-PEI based Cas9/sgRNA delivery system. Reprinted with permission from ref. . Copyright 2017 John Wiley & Sons. Inc.

Fig. 3

Non-viral vectors for in vivo CRISPR/Cas9 delivery to treat monogenic disorders. (A) Rational design of CRISPR–gold nanoparticles to deliver Cas9/sgRNA RNPs and donor DNA template to correct dystrophin gene in the mouse model of Duchenne muscular dystrophy (DMD). CRISPR–Gold was injected into the hind leg muscle of mdx mice simultaneously with cardiotoxin (CTX), which activated the proliferation of muscle stem cells by muscle damage. Two weeks later, Trichrome staining was performed on the tibialis anterior muscle to determine the levels of muscle fibrosis after different treatment. Reprinted with permission from ref. . Copyright 2015 Nature Publishing Group. (B) Rational synthesis of cationic lipids for Cas9/sgRNA RNPs delivery to ameliorate hearing loss in Beethoven (Bth) mouse model of human genetic deafness. Cas9/sgRNA–lipid complexes targeting the Tmc1^Bth allele were locally injected into the cochlea of neonatal Tmc1^Bth/+ mice. The Effects of Cas9–Tmc1-mut3 sgRNA–lipid injection on hair-cell function and hearing rescue in mice was assessed by confocal microscopy imaging and acoustic startle responses. Reprinted with permission from ref. . Copyright 2015 Nature Publishing Group.

Fig. 4

Non-viral vectors for in vivo CRISPR/Cas9 delivery to treat cancer. (A) Schematic process of RRPHC artificial virus-mediated in vivo CRISPR/Cas9 delivery for gene editing-based cancer therapy. RRPHC/Cas9-hMTH1 treatment induced ~ 42.43% frame-shift mutation of hMTH1-target loci and down-regulated the expression of MTH1 protein in SKOV3 cells. Reprinted with permission from ref. . Copyright 2017 American Chemical Society. (B) Preparation process of LACP and the schematic process of laser-enhanced knock-outs of targeted genes by LACP in A375 cells. LACP encapsulating Cas9-Plk-1 plasmid down-regulated the expression of Plk-1 protein in A375 cells with laser irradiation. Reprinted with permission from ref. . Copyright 2017 John Wiley & Sons. Inc.