CRISPR/Cas9 Protein Delivery Technologies

Progress on CRISPR/Cas9 Delivery Using Synthetic Systems

  • Fig. 1: Nanoassemblies of engineered Cas9 protein and gold nanoparticles for packaging and delivering CRISPR components into HeLa cells. a) Nanoassemblies of Cas9En, sgRNA and AuNPs, b) FITC-labeled Cas9En-RNP directly delivered into cell cytoplasm/nucleus through a membrane fusion-like mechanism. c) Indel gene editing of AAVS1 and PTEN genes in HeLa cells. (1. NP:Cas9E20-RNP; 2. Cas9E20-RNP only; 3. cells only. [Reprinted and adapted with permission from reference 18 and 28 respectively. Copyright (2017) American Chemical Society]
  • Tab. 1: Efficacy comparison of various delivery formats for the CRISPR/Cas9 system. [The information was collected from reference 18]
The CRISPR/Cas9 system has emerged as a potentially game-changing tool for gene therapy. However efficient delivery of CRISPR components in mammalian cells is required for successful genomic editing. Different delivery formats with the ability to overcome issues connected with the use of viral, plasmid and RNA strategies have been developed, with protein-based CRISPR delivery being particularly promising. This review will cover advances in delivering Cas9 protein for gene editing.
Selective interrogation and re-engineering of mammalian genes is an emerging area of opportunity [1-5]. The advent of the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated endonuclease 9) system [6] has sparked a revolution in therapeutic genomics, generating new pharmacological strategies for numerous genetically-derived diseases which, until recently, were considered virtually untreatable [7,8].
The CRISPR/Cas9 system incorporates two major components: the endonuclease protein Cas9, which has the capability to ‘cut’ specific segments of double-stranded DNA when complexed with a single-guide RNA sequence (sgRNA). This interacts with domains of the Cas9 protein and subsequently ‘guides’ the enzyme to the specific DNA sequence, allowing its repair via inherent cellular machinery [9,10].
Perhaps the largest hurdle to overcome in efficacious genomic editing is quite simply the delivery of the CRISPR components into the cell nucleus. Multiple delivery methods have been proposed to facilitate this delivery, the three most common being: i) adeno-associated plasmid/viral vectors for Cas9 and sgRNA in gene-based delivery [10-12]; ii) Cas9, mRNA, and a synthetic sgRNA in RNA-based delivery [10,13]; and iii) Cas9 protein and a synthetic sgRNA in protein-based delivery [10,14].
Viral vectors have been shown to be effective transduction agents for the delivery of the CRISPR/Cas9-mediated genome-editing system [11,15]. However, viral-based CRISPR delivery can elicit a significant immune response in vivo [16], and may cause unintentional mutagenesis of essential genes, due to incorporation into random locations in the genome [17].

Delivery of CRISPR components in plasmid format along with single stranded-DNA has been proposed to bypass the challenges associated with viral delivery [5]. However, the therapeutic potential of this delivery method is limited by both its permanence, and its high rate of off-target effects. A far more promising approach, direct delivery of the Cas9/sgRNA provides transient exposure of the host genome to the CRISPR/Cas editing system, offering higher editing efficiency, lower immunogenicity, and minimal off-targeting effects as compared to other methods of delivery (tab. 1). In this review, we will discuss some recent advances in protein delivery technologies for the CRISPR/Cas9 system.

Protein-Based CRISPR Delivery Technologies
1) Lipid-Based Systems
Lipid formulations have emerged as promising delivery vectors for CRISPR/Cas9 protein in mammalian cells, maintaining protein stability and activity. Liu et al. have demonstrated that Cas9:sgRNA  complex encapsulated into cationic liposomes can be delivered in the mouse inner ear in vivo, achieving 20% Cas9-mediated genome modification in hair cells [19]. More recently, bio-reducible disulfide bonds have been integrated into lipids to facilitate endosomal escape of lipid nanoparticles containing Cas9:sgRNA cargo, enabling genome editing with efficiencies greater than 70% [14]. However, both these systems are topically injected directly into the inner ear or brain of mice respectively, leading to efficient delivery only at the injection site. 
2) Polymer-Based Systems
Several polymer systems have also been developed for the purpose of delivering genome-editing systems in vitro and in vivo [20-24]. Polyethyleneimine (PEI) is the most common cationic polymer used for delivery of CRISPR components because structural properties, degree of branching or linearity, and molecular weight play an important role in the delivery efficiency and toxicity of the polymer [23]. Recently, Kang et al. [24] have introduced a non-viral treatment strategy by conjugation of Cas9 protein with branched PEI and delivered the genome-editing material for targeting antibiotic resistance. The nano-sized polymer-derived CRISPR/Cas9 system was designed to target mecA, the major gene involved in methicillin resistance, with potential editing of the bacterial genome. However, the gene editing efficiency was evaluated only by measuring bacterial growth after treatment with the nano-complex. Therefore, further studies are required to prove the editing efficiency of this system.
3) Cell-Penetrating Peptide-Based Systems
Cell-penetrating peptides (CPPs) have also been used to achieve efficient delivery of Cas9 and sgRNA. Ramakrishna et al. [25] reported a CPP-based delivery system in which Cas9 is conjugated to CPP while sgRNA is complexed with the peptide to form positively charged nanoparticles. This strategy directly carried Cas9 and sgRNA, enabling efficient gene editing with reduced off-target effects in human cells. However, the delivery efficiency and mechanism were not investigated. In fact, CPPs are known to undergo endosomal entrapment, resulting in low delivery efficiency [26] or high toxicity [27], while an efficient delivery into the nucleus is required for achieving successful genome editing without affecting cell homeostasis. 
4) Nanoparticle-Based Systems
As mentioned above, efficient delivery of CRISPR/Cas9 into the cytosol and nucleus is the key factor for efficient genome editing. Recently, Rotello has approached this issue by utilizing a glutamic acid ‘E-tag’ on the Cas9 complex (Cas9En) which interacts with arginine-functionalized gold-nanoparticles to form a single delivery vector [28]. This system allowed direct and efficient delivery of CRISPR/Cas9-ribonucleoprotein (Cas9-RNP) into the cytosol followed by translocation to the nucleus. In vitro studies have yielded promising results, with ~90% cytosolic and nuclear delivery in various cultured cell lines and editing efficiency of the human AAVS1 and PTEN genes in the order of ~30% (fig. 1).
In summary, delivery strategies for Cas9 are still in their infancy. Lipid formulations have shown potential for localized use in vivo, and CPPs conjugated to Cas9-RNP have shown some promise in vitro. Nano-assemblies of engineered Cas9 with gold nanoparticles are likewise promising in vitro, but need to be validated in vivo.
Support from the NIH is acknowledged (EB022641, GM077173) F. S. gratefully acknowledges FIRC (Italian Foundation for Cancer Research, Project Code: 18116) for the financial support.

Dr. Federica Scaletti1, David Luther1, Dr. Rubul Mout1, Moumita Ray1, Yi-Wei Lee1, Prof. Vincent M. Rotello1

1 University of Massachusetts, Amherst, MA, USA

Prof. Vincent M. Rotello

University of Massachusetts
Amherst, MA, USA

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