Delivering CRISPR: a review of the challenges and approaches

Abstract

Gene therapy has long held promise to correct a variety of human diseases and defects. Discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), the mechanism of the CRISPR-based prokaryotic adaptive immune system (CRISPR-associated system, Cas), and its repurposing into a potent gene editing tool has revolutionized the field of molecular biology and generated excitement for new and improved gene therapies. Additionally, the simplicity and flexibility of the CRISPR/Cas9 site-specific nuclease system has led to its widespread use in many biological research areas including development of model cell lines, discovering mechanisms of disease, identifying disease targets, development of transgene animals and plants, and transcriptional modulation. In this review, we present the brief history and basic mechanisms of the CRISPR/Cas9 system and its predecessors (ZFNs and TALENs), lessons learned from past human gene therapy efforts, and recent modifications of CRISPR/Cas9 to provide functions beyond gene editing. We introduce several factors that influence CRISPR/Cas9 efficacy which must be addressed before effective in vivo human gene therapy can be realized. The focus then turns to the most difficult barrier to potential in vivo use of CRISPR/Cas9, delivery. We detail the various cargos and delivery vehicles reported for CRISPR/Cas9, including physical delivery methods (e.g. microinjection; electroporation), viral delivery methods (e.g. adeno-associated virus (AAV); full-sized adenovirus and lentivirus), and non-viral delivery methods (e.g. liposomes; polyplexes; gold particles), and discuss their relative merits. We also examine several technologies that, while not currently reported for CRISPR/Cas9 delivery, appear to have promise in this field. The therapeutic potential of CRISPR/Cas9 is vast and will only increase as the technology and its delivery improves.

Keywords: CRISPR; Cas9; drug delivery; history; overview; prospective; review.

Figure 1.

Following formation of a double stranded break (DSB), endogenous DNA repair can occur by (A) non-homologous end joining (NHEJ) resulting in random indels, or by (B) homology-directed repair (HDR) which uses a template DNA strand for precise repair.

Figure 2.

Products of site-specific nuclease-based gene editing: (A) gene knockout, (B) gene deletion, (C) gene correction, and (D) gene addition.

Figure 3.

Site-specific endonucleases with programable DNA-binding protein domains: (A) zinc finger nucleases (ZFNs) and (B) transcription activator-like effector nucleases (TALENs).

Figure 4.

Biology of the type II CRISPR/Cas system. (A) Genomic representation of CRISPR/Cas9 along with relevant transcription/translation products. (B) Engineered CRISPR/Cas9 for site-specific gene editing (sgRNA:Cas9). Grey arrows indicate sites of single-stranded nucleotide breaks.

Figure 5.

Physical methods for delivery of CRISPR. (A) Microinjection disrupting two genes (Ppar-γ and Rag1) in Cynomolgus monkeys from a single injection into one-cell-stage embryos. Photographs of Founder Monkeys A and B, PCR products of the targeted loci from genomic DNA of A and B, and a control wild-type Cynomolgus monkey (Con). Adapted with permission from Nui et al. (2014). Copyright 2014 Elsevier Inc. (B) Electroporation delivery of CRISPR RNP targeting genes impacting mice coat color (Tyr) followed by transfer to pseudopregnant mothers. Bar plot quantifies coat color phenotypes generated from microinjection and electroporation at 1 ms pulse length and 3 ms pulse length. Adapted with permission from Chen et al. (2016). Copyright 2016 American Society for Biochemistry and Molecular Biology. (C) Hydrodynamic injection of CRISPR into mice results in liver-specific targeting (see bioluminescence image of hydrodynamically injected luciferase plasmid), generating indel mutation of two tumor suppressor genes and oncogenes. The development of liver tumors can be seen in the hematoxylin and eosin (H&E) and cytokeratin 19 (Ck19)-stained micrographs. Adapted with permission from Xue et al. (2014). Copyright 2014 Macmillan Publishers Ltd: Nature.

Figure 6.

Viral vector methods for delivery of CRISPR. (A) AAV delivery of Cas9 and sgRNAs disrupting mutations in the Dmd gene in adult mdx mice, resulting in improvement of muscle biochemistry and function. Adapted with permission from Long et al. (2016). Copyright 2016 American Association for the Advancement of Science. (B) AAV intratracheal instillation delivery of sgRNAs in Cre-dependent Cas9 knock-in mice, resulting in lung adenocarcinoma (EGFP-positive tumors). Adapted with permission from Platt et al. (2016). Copyright 2014 Elsevier Inc. (C) A split Cas9 system in which the Cas9 C-terminal is packaged into one AAV vector and the Cas9 N-terminal is packaged into a second AAV vector. Reconstitution results in a fully functioning Cas9. Reprinted from Truong et al. (2015). Copyright 2014 The Authors (CC BY license). (D) AdV delivery of Cas9 and sgRNA targeting the Pten gene in mouse liver resulting in Pten mutation (see arrows by gel), and massive hepatomegaly and features of NASH in infected livers. Immunohistochemistry shows loss of Pten staining (arrows) one month after AdV infection; H&E stained micrographs show sections of steatosis (lipid accumulation, arrows) four months post infection. Adapted with permission from Wang et al., . Copyright 2015 Mary Ann Liebert, Inc. Publishers. (E) Schematic of a lentivirus and CRISPR-based gene library functional screen used to identify the genes essential for West-Nile-virus-induced cell death. Reprinted from Ma et al. (2015). Copyright 2015 The Authors (CC BY license).

Figure 7.

Lipid nanoparticle and polyplex delivery of CRISPR. (A) Combining bioreducible lipid nanoparticles and anionic Cas9:sgRNA complexes drives the electrostatic self-assembly of nanoparticles (see TEM micrograph of 3-O14B/Cas9:sgRNA nanoparticles) for potent protein delivery and genome editing. Adapted with permission from Wang et al. (2016). Copyright 2017 National Academy of Sciences. (B) Microinjection of PEI with Cas9- and sgRNA-encoding plasmid DNA into mouse brain directed against the Ptch1 locus to generate a malignant brain tumor model. Compare the wild type and Trp53± H&E stained micrographs (arrows indicate small lesions encompassing only one cerebellar folium) with the tumor from the Trp53^−/− condition (MB = medulloblastoma). Adapted from Zuckermann et al. (2015). Copyright 2015 The Authors (CC BY license).

Figure 8.

Nanomaterial delivery vehicles for CRISPR delivery. (A) DNA ‘nanoclew’ loaded with Cas9:sgRNA RNP via Watson-Crick base pairing, followed by coating with PEI to improve endosomal escape. Reprinted with permission from Sun et al. (2015). Copyright 2015 John Wiley & Sons. (B) Arginine-modified gold nanoparticles (ArgNPs, positively charged) interact with multiple Cas9:sgRNA RNPs engineered with an E-tag to form a local negatively charged region, forming a nanoassembly that delivers Cas9 via membrane fusion. Reprinted with permission from Mout et al. (2017). Copyright 2017 American Chemical Society. (C) Synthesis of AuNPs engineered to complex with multiple Cas9:sgRNA RNPs, followed by coating in silica and the endosomal disruptive polymer PASp(DET). Adapted with permission from Lee et al. (2017). Copyright 2017 Macmillan Publishers Ltd: Nature Biomedical Engineering.