Biomaterials as vectors for the delivery of CRISPR–Cas9

Joon Eoh and Luo Gu *
Department of Materials Science and Engineering, Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, Maryland 21218, USA. E-mail: luogu@jhu.edu

Received 16th October 2018 , Accepted 25th January 2019

First published on 8th February 2019


The emergence of the CRISPR–Cas9 gene editing system has brought much hope and excitement to the field of gene therapy and the larger scientific community. However, in order for CRISPR-based therapies to be translated to the clinical setting, there is an urgent need to develop optimized vectors for their delivery. The delivery vector is a crucial determinant of the therapeutic efficacy of gene editing and should be designed to accommodate various factors including the form of the payload, the physiological environment, and the potential immune responses. Recently, biomaterials have become an attractive option for the delivery of Cas9 due to their tunability, biocompatibility and increasing efficacy at drug delivery. Biomaterials offer a unique solution for creating tailored vectors to meet the demands of various applications that cannot be easily met by other delivery methods. In this review, we will discuss the various biomaterial systems that have been used to deliver Cas9 in its plasmid, mRNA and protein forms. In addition, the functions of these materials will be reviewed to understand their roles in Cas9 delivery. Finally, the immune challenges associated with Cas9 and the delivery materials will be discussed as an understanding of the immune responses along with the functions of biomaterials will ultimately guide the field in designing new delivery systems for the clinical applications of CRISPR–Cas9.


1. Introduction

The advent of gene editing platforms such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the CRISPR–Cas9 system has significantly impacted the scientific community in the past decade, bringing about numerous advances in fields such as gene therapy, developmental biology, cancer research, etc. In particular, the simplicity and versatility of the CRISPR–Cas9 system has raised hopes that genetic disorders, once thought to be incurable, may be treated in the near future. The CRISPR–Cas9 system consists of Cas9 endonuclease, a single guide RNA (sgRNA), and if applicable, a donor template for homologous repair (Fig. 1a). The same Cas9 can be used with different sgRNA and donor templates, offering flexibility for various applications.1–5 The versatility of the system further extends to the Cas9 nuclease being able to be delivered as DNA, mRNA or protein.4,6 There are advantages and disadvantages to each approach and they will be further discussed in this review. Despite its great potential, there are still a number of biological hurdles that must be addressed in order for CRISPR–Cas9 to be used broadly for therapeutic applications. Extracellular challenges include clearance by the mononuclear phagocyte system, nuclease or protease cleavage, issues with tissue specificity, and immune complications arising from innate and adaptive immune responses.7 At the target cells, nanoparticles carrying the CRISPR–Cas9 cargo face intracellular challenges such as traversing through both the cell and nuclear membranes, as well as endosomal escape to maintain the therapeutic efficacy of the CRISPR–Cas9 system.7 In light of these challenges, it is necessary to further investigate the delivery methods used for the CRISPR–Cas9 components in order to improve their efficiency of gene editing.
image file: c8bm01310a-f1.tif
Fig. 1 Biomaterials used for the delivery of different forms of Cas9. (a) The Cas9 endonuclease binds to a sgRNA, which allows specific editing of a target DNA sequence next to a protospacer adjacent motif (PAM). At the target sequence, Cas9 undergoes a conformational change resulting in cleavage of both strands. The most common method of repair is non-homologous end joining (NHEJ), but incorporating a donor template allows for sequence-specific homology directed repair (HDR). (b) Cas9 can be delivered in the form of a plasmid,2 mRNA, or protein.138 For the delivery of a plasmid, liposomes,32,51,52,58,59,64–66 PEI,34,37 and exosomes50,51 are some of the most commonly used vectors. For Cas9 mRNA, liposomes have been primarily used,12,72,73,77 but other biomaterials such as amino lipid nanoparticles78,80 and extracellular vesicles82 have also been used. For Cas9 in the protein form, liposomes,91,95,98,104 Cas9-peptide complexes96,97 and gold nanoparticles104,105,112 are some of the commonly used delivery systems.

Current delivery strategies range from physical methods such as electroporation and microinjection to delivery via viral and/or non-viral vectors. Physical methods often subject cells to temporary stimuli causing perturbations in the membrane, providing a small time frame for improved delivery across biological membranes.4 Methods such as microinjection even allow for the precise and selective gene editing of single cells. Despite their success in vitro,8–11 more work must be done for optimizing physical methods for in vivo applications.

The use of viruses, namely integration-deficient lentivirus, adenovirus and adeno-associated virus, is one of the most effective and commonly used methods for delivering the components of CRISPR–Cas9. The advantages of these vectors include a high transfection rate and diverse tropism owing to the different serotypes of the adeno-associated virus (AAV).7 Viral vectors have been successfully utilized in vivo in various disease models including Duchenne muscular dystrophy, hereditary tyrosinemia type I and retinitis pigmentosa.12–16 However, there are drawbacks to using viral vectors. In particular, the physical capacity of viral vectors is often a challenge for CRISPR delivery as the maximum capacity of an AAV is approximately 4.7 kb. As such, separate vectors are often needed to deliver the various components.4,17 Tropism becomes an issue if specific targeting of an organ system is desired as there may be multiple systems targeted for a given serotype.7 In addition, retroviral vectors have been associated with increased incidence of leukemia, and the prolonged duration of expression for the adenoviruses and AAVs warrants further consideration as increased exposure may lead to unintended gene editing in other organ systems due to the overlapping tropism of viral serotypes.7,18–20 In the case of multiple doses, viral delivery is less ideal due to the immunogenicity of the adenoviruses and the increased risk of an adaptive immune response to the AAV capsids.7,21

The application of biomaterials as non-viral vectors has gained popularity in recent years due to their versatility, biocompatibility and increasing transfection efficiency. Capacity limitations are no longer an issue and nanoparticles can be further customized to improve tissue specificity as well as nuclear transport.4 This tunability aspect of biomaterials holds great promise in optimizing the delivery of CRISPR–Cas9 for in vivo gene editing. Because biomaterials have virtually limitless options for customization, they provide a distinct advantage to physical and viral delivery since different cell types and organ systems will ultimately require gene editing strategies tailored to meet the demands of their microenvironments.

A number of excellent reviews have been written on the delivery of Cas9.4–7,22–24 In this review, we focus on providing concise analyses of studies that reflect a rapidly progressing trend in using biomaterials for delivering Cas9 in its DNA, mRNA or protein form (Fig. 1b). A great emphasis is placed on discussing the various biomaterial components and their overall contributions to the delivery process. A comprehensive list of materials used for Cas9 delivery and the functions of commonly used biomaterials are also included in this review (Tables 1 and 2). Another emphasis of this review is the immune responses to the delivery materials and Cas9 itself. There is an increasing understanding that the immune system is often a major challenge for many drug delivery systems. Because of its bacterial origin, innate and adaptive immune responses should also be considered when using the Cas9 nuclease.21,25,26 As the field continues to work on expanding the efficiency and complexity of Cas9 delivery systems, the immune responses to biomaterials and the Cas9 system should be an important point of focus. This review will describe in further detail some of these immune related concerns regarding the use of biomaterials and Cas9 in its various forms.

Table 1 Cas9 cargoes and the biomaterial vectors used to deliver them
image file: c8bm01310a-u1.tif


Table 2 Common components of Cas9 delivery systems and their functions
image file: c8bm01310a-u3.tif


2. Delivery of Cas9 in DNA form

The most commonly used form of Cas9 DNA is derived from Streptococcus pyogenes (SpCas9) and its plasmid is approximately 4.2 kb,27 while another commonly used form is derived from Staphylococcus aureus (SaCas9) at about 3.2 kb.28 As each species and variant recognizes different PAMs,28–30 this allows for greater accessibility of gene editing. Using the plasmid DNA form of the Cas9 nuclease is advantageous due to its low cost, stability, as well as the potential for prolonged production of the Cas9 nuclease for continuous gene editing.4,6 A typical plasmid consists of sequences encoding the Cas9 nuclease, nuclear localization signals (NLS) to allow for nuclear shuttling, a promoter to begin transcription, and sometimes sgRNA (Fig. 1b). Because naked plasmid DNA cannot pass through the cell membrane due to its hydrophilicity, large size, and negative charge, carrier particles are often employed in order to provide a net positive charge for easier passage across the membrane and protection from enzymatic degradation.31

2.1 Expanding upon the conventional nanoparticles for transfection

Many cationic lipid- and polymer-based delivery systems are commercially available through transfection reagents such as Lipofectamine (cationic lipid) and Turbofect (cationic polymer). Such products have even been used in vivo as shown by Moutal et al., where Turbofect was used to encapsulate a plasmid encoding Cas9, GFP for measuring transfection efficiency, a sgRNA targeting neurofibromatosis 1 (Nf1) for truncation.32 Localized, repeated intrathecal delivery through implanted catheters in Sprague-Dawley rats resulted in on-target editing of the Nf1 gene and increased nociceptive activity, consistent with the symptoms of neurofibromatosis type 1. This use of CRISPR–Cas9 allowed the authors to identify cytosolic regulatory protein collapsin response mediator protein 2 (CRMP2) as a therapeutic target for treating the symptoms of NF1-related pain.32

Despite the ease of use and convenience of commercial transfection reagents, studies such as the one conducted by Zhang et al. suggested several drawbacks of these products such as the limitations of carrying large size Cas9–sgRNA plasmids and insufficient shielding of the plasmid's negative surface charge.33 To address these issues, the Cas9–sgRNA plasmid targeting the gene encoding polo-like kinase 1 (PLK-1) was complexed with chondroitin sulfate and then protamine, which formed a tightly bound core with a net negative charge. The core was then encapsulated with a cationic lipid mixture of DOTAP, DOPE and cholesterol. These nanoparticles demonstrated increased transfection efficiency both in vitro and in vivo when compared with commercial transfection reagents. Repeated intratumoral injection of the nanoparticles caused inhibited A375-tumor growth, likely due to knockout of PLK-1 in some of the cancer cells. While there were initial concerns about cytotoxicity of the lipids and nonspecific interaction with serum proteins, surface modification of the nanoparticles with DSPE-PEG mitigated this problem, demonstrating the advantage of tunability when using biomaterials as delivery vehicles.33

Yan et al. were able to demonstrate another unique solution for working around the large size of the CRISPR–Cas9 system using previously established DNA cassette nanocapsules adapted for CRISPR–Cas9 delivery.34 DNA cassettes encoding sgRNA were encapsulated with nanocapsules composed of a hydrophilic monomer (tris-acrylamide), a positively-charged monomer (acryl-spermine), a non-degradable cross-linker (N,N′-methylenesbisacrylamide, BIS) and a degradable cross-linker (glycerol 1,3-diglycerolate diacrylate, GDGDA). The Cas9 plasmid was introduced by condensing with dioleoylphosphatidylethanolamine-conjugated, branched polyethylenimine (PEI-PE). The nanocapsule assembly began with the hydrophilic and positively charged monomers complexing with the DNA cassette, where in situ polymerization then resulted in a nanocapsule that was stable at physiological pH. However, the nanocapsule was able to disassemble and undergo endosomal escape at a lower pH due to the proton sponge effect. The PEI-Cas9 and sgRNA nanocapsule co-transfection system was found to knock out EGFP expression by about 30% in CEM T-cells.34

PEI is one of the most commonly used polymers for intracellular delivery due to its high transfection efficiency and ability to aid in endosomal escape. This is mainly attributed to its high density of amine groups.35,36 These positively charged amine groups also allow for increased interactions with nucleic acids. Ryu et al. designed a delivery system using only branched PEI.37 As the cytotoxicity of PEI is dependent on its molecular weight, a validated molecular weight of 25 kDa PEI was chosen for the study. The use of only branched PEI allowed for quick and simple polyplex formation at room temperature. Varying ratios of PEI to plasmid were used to study complex formation, toxicity, and transfection efficiency. In order to assess the gene editing efficiency of the PEI delivery system, Cas9 plasmid co-expressing Cas9 and sgRNA targeting the Slc26a4 gene was used. Polyplexes formed with an optimal ratio (N/P 15) of branched PEI and Cas9 plasmid were found to have a similar gene editing efficiency to Lipofectamine 2000 in Neuro2a cells.

Altering the physical flexibility of nanoparticles has been shown as one strategy to enhance transfection efficiency as well as the packaging capacity. Kretzmann et al. took advantage of the unique molecular structure and properties of dendrimers to improve the delivery of large plasmid DNA.38 Poly(amido amine) (PAMAM) dendrimers were used as the high density of primary amines allowed for interactions with the plasmid to form stable polyplexes while the high density of tertiary amines provided buffering capacity for endosomal escape. The PAMAM dendrons were anchored on a linear copolymer of poly(2-hydroxyethyl methacrylate) (HEMA) and glycidyl methacrylate (GMA). The optimum dendron density and dendron generation were determined in order to prevent significant non-specific interaction and cytotoxicity.38 The polymers were then fluorinated as it has been shown that fluorination enhances cellular uptake, facilitates endosomal escape and provides stability in the serum.39 Compared with Lipofectamine 2000, the fluorinated dendronized polymer more effectively transfected MCF-7 cells and achieved significantly higher MASPIN expression using a sgRNA-guided dCas9 gene activation system.38

Incorporation of stimuli-responsive components into nanoparticles would allow for spatial and temporal control over the release of the Cas9 plasmid. Qi et al. recently develop a pH-responsive Cas9 delivery system using an acid-labile polycation decorated with fluorinated alky chains (ARP-F).40 Positively charged ARP-F can condense negatively charged plasmids to form stable nanoparticles by electrostatic interaction. Besides the fluorinated alky chains that help to across the cell and endosome membranes,39 the hydroxyl groups on ARP-F provide additional hydrophilicity for blood compatibility. Transfection measured through reporter plasmids revealed comparable transfection efficiency between ARP-F vector and PEI vector in both HEK 293 and A549 cells. However, cell viability was much higher for ARP-F treated cells in comparison with PEI. It was found that the acid-labile ortho ester groups in ARP-F promoted the release of plasmid payload in cells due to their degradation by the acidic endosomes. In addition, the non-acid-labile polycation exhibited decreased transfection efficiency, showing that the degradable moieties of ARP-F vector benefited its transfection performance.

ARP-F was then used to deliver a Cas9 plasmid encoding Cas9, a sgRNA targeting the apoptosis inhibitor Survivin, and GFP for measuring transfection efficiency. Successful indel formation in the Survivin gene and a reduction of Survivin protein level were observed in A549 cells transfected with the ARP-F/Cas9 vector. Additional in vivo studies using the ARP-F/Cas9 vector via tail vein injections were also performed on A549 tumor-bearing BALB/c nude mice. Overall, mice treated with the ARP-F/Cas9 vector in combination with the anti-cancer drug temozolomide had the lowest tumor volume with reduced Survivin protein expression.

The use of core–shell principles was also demonstrated in a study conducted by Timin et al.41 Hollow hybrid microparticles composed of poly-L-arginine and dextran sulfate were encased within a silica shell and used to deliver CRISPR–Cas9 components. Poly-L-arginine and dextran sulfate capsules were chosen as they have been shown to successfully deliver nucleic acids to the cytosol of target cells.42,43 The SiO2 shell may provide additional colloidal stability and protection of the payload bioactivity. In this study, both the SiO2 coated polymer capsules and the non-coated capsules were more efficient at editing a model fluorescent protein gene in HEK293T cells than a commercial liposome-based transfection reagent.41

Micelles using only 3 components have also been used for the delivery of condensed plasmid Cas9–sgRNA as shown by Lao et al. An optimized blend of quaternary ammonium-terminated poly(propylene oxide) (PPO-NMe3) and amphiphilic Pluronic F127 was used to deliver the system for targeting the HPV18-E7 oncogene in a human papillomavirus (HPV) model.44 It was hypothesized that too high a charge density would have a negative effect on plasmid delivery due to the large size of the plasmid and the potential long-term effects of Cas9 expression. Therefore, PPO-NMe3 was chosen to condense the plasmid rather than PPO-NH2 due to the stronger nucleic acid-binding affinity of quaternary ammonium without increasing charge density. F127 was then used to improve colloidal stability and transfection efficiency. Micelles then formed from electrostatic and hydrophobic interactions of the 3 components.44 The results demonstrated disrupted viral oncogene expression in vitro through sustained Cas9 activity, and in vivo results showed reduced tumor volume and increased expression of the tumor suppressor, Rb.44

Improved transfection efficiency and reduced cytotoxicity have been shown by Dharmalingam et al. upon altering the molecular architecture of nanoparticles using fatty acyl chains from palmstearin.45 Although the authors previously found that hydrophobic chain asymmetry could enhance transfection using coconut oil, palmstearin was used in this study due to the minimal variability in fatty acyl chains. Even though palmstearin-based nanoparticles with an asymmetric hydrophobic core in the lipids were able to deliver the components of CRISPR–Cas9 better than the symmetric controls, neither were shown to be able to transfect HEK-293 cells better than the control Lipofectamine 3000.45 Despite this, the authors are able to still demonstrate that asymmetry in the molecular architecture can play a significant role in the delivery of nucleic acids.

The use of cell penetrating peptides (CPPs) could be an important strategy to consider when designing a delivery vehicle, as they may better facilitate cell entry and endosomal escape. While cell penetrating peptides alone have been used to deliver CRISPR–Cas9,46,47 the stand-alone use of these peptides does not effectively protect the components from degradation. Rather, the ideal use of CPPs may be as a supplement to various nanoparticles. The helical, cationic polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-L-glutamate) was used in a study by Wang HX et al. due to its ability to condense plasmids while retaining the helical structure necessary to penetrate the membrane for internalization and endosomal escape.48 The polypeptide is also robust in the physiological environment, and this stability is believed to be due to its non-natural amino acid sequences that are not recognized by proteases. To further enhance its stability for in vivo use, polyethylene glycol (PEG) was incorporated into the helical polypeptide nanoparticles to prevent non-specific protein adsorption. To evaluate the helical polypeptide nanoparticles as Cas9 plasmid delivery vehicles, Cas9-GFP plasmid transfection was performed in a variety of cell types. Cas9-GFP expression was significantly higher when using these nanoparticles in comparison with expression using PEI conjugation and, in some cell lines, expression using Lipofectamine 3000.48In vivo experiments targeting PLK-1 in an A549.GFP tumor model showed reduced expression of PLK-1 as well as tumor growth inhibition and increased animal survival.48

Exosomes are cell secreted carrier particles that carry proteins, lipids and nucleic acids and can stably exist under physiological conditions. As such, they are an attractive alternative to synthetic nanoparticles due to their biocompatibility and specific cell-targeting capabilities.49 In a recent study by Kim et al., exosomes derived from SKOV3 ovarian cancer cells were loaded with the Cas9/sgRNA plasmids targeting PARP-1 via electroporation.50 PARP-1 was chosen as a target due to its clinical promise in BRCA1/2 deficient breast and ovarian cancers. Mice bearing SKOV3 xenograft tumors were administered with the PARP-1 targeting Cas9/sgRNA exosomes either intravenously or intratumorally. Reduced expression of PARP-1 and tumor growth inhibition were detected, indicating the anti-tumor effects of the Cas9/sgRNA loaded exosomes. It was also found that the tumor uptake efficiency was higher for the SKOV3-derived exosomes than for HEK293-derived exosomes, suggesting specific tropism according to cell origin. However, tumor uptake was still detected in the HEK293 exosomes, which suggests the need for further investigations on how tropism in exosomes is determined. Liposome and exosome hybrid particles have also been used to better facilitate the larger size of Cas9 plasmid. By incubating HEK293FT-derived exosomes with a mixture of Lipofectamine 2000 and the Cas9–sgRNA plasmid targeting a model gene, CTNNB1, a hybrid exosome system was created that could cleave the CTNNB1 gene in mesenchymal stem cells.51

2.2 Addressing tropism

Tissue and cell specificities are much desired in gene editing, as this can reduce required dosages and decrease off-target and other adverse effects. One strategy is to customize plasmids with a cell-specific promoter that allows for selective gene editing only in certain types of cells. For example, CD68 proteins are expressed specifically in monocytes and macrophages. This observation was used in a recent study that combined the CD68 promoter and a sgRNA sequence targeting the Ntn1 gene in one plasmid to allow for targeted gene disruption in monocytes and macrophages for possible type 2 diabetes treatment.52 The authors used a cationic lipid N,N-bis(2-hydroxyethyl)e-N-methyl-N-(2-cholesteryoxycarbonyl-aminoethyl) ammonium bromide (BHEM-Chol)-assisted PEG-b-PLGA nanoparticle (CLAN) system53 to encapsulate the large plasmid via a double emulsion method. The cationic lipid assisted with plasmid encapsulation, and the presence of the nanoparticles, specifically the hydrophilic PEG, provided steric stabilization and prevented aggregation and protein adsorption.52–54 Even though the nanoparticles were endocytosed in other cell types, netrin-1 (Ntn1) expression was only disrupted in macrophages and monocytes due to the macrophage-specific promoter.52

As some cells may possess unique signatures in their genome such as the BCR-ABL fusion gene region in chronic myeloid leukemia (CML), these features can be exploited for cell-specific gene editing. Liu et al. encapsulated Cas9–sgRNA plasmid targeting the overhanging fusion region of the BCR-ABL gene in K-562 CML cells using the same cationic lipid-assisted PEG-b-PLGA system (CLAN) as that used in the previously mentioned study.55 Using a CML mouse model, it was found that the intravenously injected nanoparticles were able to reduce mRNA and protein expression of BCR-ABL, resulting in enhanced survival in mice.

Certain surface receptors such as the folate receptor are often overexpressed in tumors,56 so using nanoparticles that target those receptors as carriers for Cas9 is another strategy to potentially increase cell-specificity in gene editing.57 In a recent study, He et al. developed a folate receptor-targeting liposome to deliver the plasmid co-expressing Cas9 and sgRNA targeting cancer-related DNA methyltransferase 1 (DNMT1) to SKOV-3 ovarian cancer cells.58 The liposomes were composed of DOTAP, Chol, and methoxy-PEG-succinyl-Chol for the prevention of nonspecific adsorption, as well as folate-PEG-succinyl-Chol for folate receptor binding. Using a xenograft ovarian cancer mouse model, it was shown that the folate modified liposomes were able to reduce DNMT1 expression and inhibit tumor growth when administered intraperitoneally.58 Future work on detailed comparison between receptor-targeting nanoparticles and non-targeting nanoparticles will help to better establish this strategy for improving cell specificity in gene editing.

Using a similar strategy, Liang et al. screened an osteosarcoma cell-specific aptamer and functionalized it to PEG-PEI-cholesterol lipopolymers encapsulating a plasmid encoding Cas9 and a sgRNA targeting VEGFA, which is known to heavily contribute to angiogenesis and cell survival within the tumor microenvironment.59 The aptamer functionalized lipopolymer nanoparticles displayed improved tumor targeting in a syngeneic orthotopic osteosarcoma mouse model (K7M2 cells) as compared to particles functionalized with an aptamer of random sequence. Furthermore, mice treated with the osteosarcoma targeting Cas9–sgRNA nanoparticles showed a reduction of tumor VEGFA as well as inhibition of tumor growth and metastasis, while Cas9–sgRNA nanoparticles with the control aptamer had little effect.59

Endosomolytic peptides can be used, in addition to aptamers, to further aid in cellular uptake and endosomal escape. Liu, B. et al. were able to develop such a system by first complexing protamine sulfate with the Cas9 plasmid. The endosomolytic peptide KALA was then incorporated into the structure along with carboxymethyl chitosan containing the aptamer AS1411.60 AS1411 has been shown to target nucleolin which is overexpressed on the surface of certain cancer cells. Initial transfection studies were performed on MCF-7 breast cancer cells using labeled plasmids. It was found that cells treated with nanoparticles containing both the aptamer and the endosomolytic peptide showed the highest plasmid uptake and expression. Delivery of the Cas9 plasmid encoding Cas9 and the sgRNA targeting the CDK11 gene in MCF-7 cells resulted in reduced protein levels of CDK11. In particular, nanoparticles containing both the aptamer and the endosomolytic peptide showed the highest gene editing efficiency with ∼21% indel frequency in the CDK11 gene. In comparison, Lipofectamine 2000 showed substantially lower editing efficiency. VEGF and matrix metalloproteinase-9 (MMP-9) down regulation and cell growth inhibition were also observed as a result of CDK11 disruption.

A dual-receptor targeting approach was employed by Li et al., who used an intriguing core–shell “artificial virus” to target ovarian cancer.61 A partially fluorinated PEI polymer (PF33) was used to complex with the plasmid encoding Cas9 and the sgRNA targeting the gene MTH1. This gene is overexpressed in many types of cancer, and knockdown of the gene would result in genomic DNA damage and apoptosis. The PF33/plasmid core was then complexed with a multifunctional shell composed of RGD-R8-PEG-HA to form a core–shell artificial virus. The RGD-R8 tandem allowed for αvβ3 integrin (and potentially other integrins) targeting, which is often overexpressed on tumor vascular endothelial cells and tumor cells, as well as a high cell penetrating capability due to the octa-arginine peptide. Hyaluronan (HA) can specifically bind to CD44, which is highly expressed on many cancer cells and also regulates metastasis;62 while PEG conjugated to the HA backbone allowed for stabilization of the artificial virus.61 Upon internalization and degradation of the shell, the positively charged PF33/plasmid complex allowed for efficient endosomal escape and nuclear localization. The artificial virus was able to transfect SKOV3 cancer cells at a higher efficiency than other commercial transfection reagents such as PEI and Lipofectamine. Effective tumor accumulation, MTH1 disruption, and tumor growth inhibition were observed in mice bearing metastatic SKOV3 tumors when treated with the core–shell artificial virus particles.61

In some cases, the physical properties of the cells themselves can affect biodistribution as well as cellular uptake as different cells exhibit different preferences for nanoparticle uptake.35,63 Liu et al. prepared a library of cationic lipid-assisted PEG-b-PLGA nanoparticles (CLAN) of varying polymer compositions and surface charge to screen for the optimal combination for delivering the CRISPR–Cas9 system to neutrophils in the epididymal white adipose tissue and the liver.64 These optimized nanoparticles would then target neutrophils in those tissues and disrupt the secretion of neutrophil elastase as a potential treatment for type 2 diabetes by reducing chronic inflammation. PEG5K-b-PLGA11K, PLGA8K and the cationic lipid BHEM-Chol were used to create the nanoparticles. Nanoparticle surface PEG density and surface charge were adjusted through varying the quantity of PEG-b-PLGA and BHEM-Chol in order to control the nanoparticles’ in vivo behavior. It was found that the optimized nanoparticles were able to effectively deliver Cas9/sgRNA to the epididymal white adipose tissue and the liver of high-fat diet-induced type 2 diabetes mice after intravenous injection and disrupted neutrophil elastase expression in those tissues.64

The use of external energy to trigger the release of CRISPR–Cas9 components provides a distinct method for spatially and temporally controlled gene editing. In a recent study, Wang, P. et al. sought to combine the unique photothermal properties of gold nanoparticles (AuNPs) with the conventional merits of lipid-based particles.65 Cationic AuNPs were created by linking cell-penetrating TAT peptides onto the AuNPs. The cationic AuNPs were then complexed with the Cas9–sgRNA plasmid targeting PLK-1. A cationic lipid formulation composed of DOTAP, DOPE, and cholesterol was then used to coat the AuNPs-plasmid and the particles were further modified with PEG2000-DSPE to improve stability. Using these AuNPs, it was demonstrated that the light irradiated nanoparticles were more effective at disrupting PLK-1 in melanoma cells than the same nanoparticles without irradiation due to photothermal energy triggered lysosome burst and release of Cas9–sgRNA plasmids. In vivo experiments also showed that the combination of the nanoparticles and photo irradiation was more effective at inhibiting tumor growth than the nanoparticles without irradiation or irradiated nanoparticles carrying a control sgRNA.65

2.3 Homology directed repair

While examples have been shown for the role of CRISPR–Cas9 delivery in gene cutting, the ability to correct mutations via homology directed repair (HDR) is more challenging, but is an area of great interest. Schu et al. have shown the potential of delivering the CRISPR/Cas9 plasmid and the correct ssDNA template for the mutated IDUA gene to treat mucopolysaccharidosis type I (MPS I) using an in vitro model. Lipid based nanoemulsions containing medium chain triglycerides (MCT), phospholipid DOPE, DOTAP for plasmid complexing, DSPE-PEG to prevent non-specific adsorption, and water were used to form the delivery system.54,66 The nanoemulsions were made using high pressure homogenization with the plasmid/oligonucleotides from the CRISPR–Cas9 system either directly adsorbed onto the nanoemulsions via the DOTAP, or complexed with DOTAP, and then encapsulated. Both formulations showed the ability to treat MPS I conditions via increased IDUA activity and reduction of lysosomes in primary MPS I fibroblasts in culture. Further work on analyzing the HDR efficiency would provide a better understanding of the delivery strategy.66

2.4 Immune-related concerns of Cas9 plasmid delivery

The immune response has always been a challenge for the delivery of conventional drugs and gene therapies,67 so naturally, the immune response to CRISPR/Cas9 based therapies is also a concern (Fig. 3). While there has not been any report of immune complications when using the plasmid form of the Cas9 nuclease, there is still the possibility of an innate immune response due to the activation of pattern recognition receptors (PRRs).68 The unmethylated CpG motif, abundant in many pathogen genomes, is known to trigger an immune response through a Toll-like receptor (TLR) pathway, making the origin of the plasmid DNA an important consideration.68,69 In addition, the presence of plasmid DNA in uncommon areas of the cell such as the cytoplasm has been shown to trigger a potent immune response via the STING-cGAS pathway.68,70 Future studies aiming at optimizing Cas9 plasmid delivery could consider designing and even modifying the plasmid itself to reduce innate immune response71 as well as exploring ways of accelerating the nucleocytoplasmic shuttling process to minimize activation of the innate immunity.

2.5 Common characteristics of materials used for Cas9 plasmid delivery

When designing a delivery system for the Cas9 plasmid, the physical properties of the plasmid are a key consideration. Due to its large size and net negative charge, most of the delivery systems incorporate components with high positive charge densities to condense the plasmid and alter its net charge for favorable interactions with the cell membrane. This high positive charge density can also aid in endosomal escape in certain cases. Because of this, many of the discussed vectors are composed of cationic lipids or PEI-based polymers (Fig. 1b). The high tunability of biomaterials allows for their physical properties to be adjusted to accommodate the large plasmid. This ability to tailor the properties of the biomaterials makes size constraints less of an issue and gives them a distinct advantage over viral vectors. Another consideration to be made is the need to deliver the plasmid to the cell nucleus. As entry through the nuclear membrane is required for the expression of the plasmid, including NLS is a common strategy when designing the delivery system.

3. Delivery of Cas9 in mRNA form

The use of Cas9 in its mRNA form offers several advantages over plasmid DNA, namely avoiding the need to access the cell nucleus, leading to quick and transient Cas9 expression.4,6 As Cas9 mRNA only needs to be delivered into the cytosol in order for expression to occur, risks associated with using Cas9 DNA such as off-target effects and integration are reduced or avoided.6 However, its instability and susceptibility to degradation5 make the development of a proper vector to protect its therapeutic viability of great importance. Several studies have already used biomaterial systems for the delivery of CRISPR–Cas9 via mRNA and will be discussed in this section.

3.1 mRNA: lost in translation

Yin et al. demonstrated one of the first applications of CRISPR–Cas9 mRNA delivery through a combined viral and non-viral approach.12 In order to work around the size constraints of Cas9 and its potential long-term adverse effects, lipid nanoparticles composed of lipid-like C12-200 molecules, cholesterol, C14PEG2000, DOPE, and arachidonic acid were used to encapsulate Cas9 mRNA through a microfluidic method. The sgRNA and HDR template, however, were delivered using AAVs. The viral and non-viral particles were then delivered to a mouse model of human hereditary tyrosinemia (Fah mutation) through intravenous injection. The correction of mutated Fah gene of hepatocytes in the mouse liver was greater than 6%.12 The viral and non-viral combination provided a strategy to deliver all the required components to achieve HDR in vivo. In addition, using biomaterials and the mRNA form of Cas9 to circumvent the size constraint of the AAV vector brings into consideration the feasibility of combining all components into one carrier. This would potentially allow for a simplified approach as well as a lower dose being needed to achieve a therapeutic effect. A follow-up study by the team continued to improve the therapeutic efficacy of the CRISPR–Cas9 system in vivo by modifying the sgRNA to reduce its susceptibility to nuclease degradation.72 By modifying with 2′ hydroxyl groups and phosphorothioate bonds, enhanced sgRNAs (e-sgRNAs) were created. Co-delivery of the e-sgRNA targeting Pcsk9 and Cas9 mRNA using the same lipid nanoparticle system as that used in the previous study exhibited significantly higher indel formation at the Pcsk9 locus in the mouse liver compared to using non-modified sgRNA.72 Using e-sgRNA targeting Fah or Rosa26 genes showed a similar enhancement.72 As the timing between dosages of Cas9 mRNA and sgRNA is a concern for efficiency and is largely based on empirical data, modification of the sgRNA as shown would allow for efficient delivery of Cas9 mRNA and sgRNA in one vector.

Finn et al. were also able to identify structural modifications to sgRNA (2′-OMe and phosphorothioate linkage) that allowed for sustainable levels of in vivo gene editing as a potential treatment for transthyretin (TTR) amyloidosis. A lipid nanoparticle system, composed of a synthetic ionizable lipid (LP01), cholesterol, DSPC, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), was used to encapsulate both the Cas9 mRNA and the modified guide RNA.73 The use of Cas9 mRNA and highly modified sgRNA targeting the Ttr gene demonstrated significantly improved gene editing in a dose dependent manner in comparison with slightly modified and non-modified sgRNA. Remarkably, this study highlights the durability of the CRISPR–Cas9 mediated effects. The percentage of edited cells in the liver continued to increase up to 180 hours, and TTR levels remained low for 12 months from just a single intravenous administration.73

Li et al. designed N1,N3,N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (TT) derived lipid-like nanoparticles74 based on previous studies detailing the required structural features for effective siRNA delivery systems.75,76 An orthogonal experimental design later found TT3 to be a highly effective polymer for in vivo delivery of mRNA,74 which was then used in a different study by Jiang et al. to deliver Cas9 mRNA and sgRNA targeting the Pcsk9 gene for treatment of hypercholesterolemia. Using an optimized formulation of TT3, DOPE, cholesterol and DMG-PEG, nanoparticles carrying the Cas9 mRNA and Pcsk9 sgRNA were injected into mice intravenously. Cas9 expression was detectable after 6 hours and became undetectable by 24 hours, suggesting a transient response.77 Furthermore, PCSK9 protein levels from liver lysates were reduced by ∼40% in a single treatment. The authors then tested the efficacy of their system on HBV associated covalently closed circular DNA (cccDNA), a major motif of the viral genome in infected hepatocytes. Intravenous injection of the TT3 nanoparticles with Cas9 mRNA and cccDNA-targeting sgRNA effectively cut HBV DNA and reduced viral load in a mouse model of HBV replication.77 An important aspect of this study is the timing between administration of the Cas9 mRNA and sgRNA. As 6 hours was the period when Cas9 was most highly expressed, the sgRNA was subsequently administered so that the CRISPR–Cas9 complex could readily form in situ rather than wait for translation of the mRNA to occur first.

Zwitterionic amino lipids (ZALs) are another interesting class of materials that possess the capability of encapsulating long sequences of RNA including mRNA and sgRNAs. This could allow for the simultaneous delivery of both components if desired. Miller et al. designed new zwitterionic amino lipids that combined the benefits of cationic and zwitterionic lipids in one system, as highly successful lipid nanoparticles often use cationic lipids to bind RNAs and promote endosomal escape while zwitterionic phospholipids aid in solubilizing RNAs.78 A balance of each component is important for maintaining the ideal intermolecular interactions, and ZALs were hypothesized to increase molecular interactions between long RNA, cationic lipid, and the zwitterionic motif in one system. Co-delivery of Cas9 mRNA and sgRNA targeting LoxP through intravenous injection in mice resulted in successful cutting of a Lox-stop cassette and induction of subsequent tdTomato expression in the liver, kidneys and lung. An important result of this study is that even though it was found that temporally separating the delivery of Cas9 mRNA and the sgRNA delivery was an effective method much like the other studies described, co-delivery was made feasible by varying the Cas9 mRNA[thin space (1/6-em)]:[thin space (1/6-em)]sgRNA ratio.78

3.2 Biodegradable carriers to reduce toxicity

Biodegradability is an important feature of nanoparticles for in vivo applications as accumulation of non-biodegradable lipids has been shown to cause adverse effects or even acute mortality in animal models.79 Because of such a consideration, Zhang, X. et al. developed nanoparticles with tunable degradability based on the configuration of the ester chains as well as the steric effects of functional groups on the lipid-like compounds that were used to formulate the nanoparticles.80 Using the core molecule N-methyl-1,3-propanediamine (MPA) and conjugating different types of lipid chains, it was found that 9-oxononanoic acid (Z)-non-2-en-yl ester (A) and 9-oxononanoic acid 2-ethyl-hexane-1-yl ester (Ab) exhibited good degradability. The amino ester lipid compounds were then transformed into nanoparticles using DOPE, cholesterol, and DMG-PEG. The resulting nanoparticles demonstrated efficient Cas9 mRNA encapsulation and transfection. In addition, intravenously injected particles showed fast clearance and little toxicity in the major organs.80

3.3 Extracellular vesicles for Cas9 mRNA delivery

Extracellular vesicles (EVs), which encompass exosomes as well as other membrane secretions,81 have also been used to deliver Cas9 in its mRNA form. Recently, Usman et al. derived EVs from red blood cells (RBC) for delivering CRISPR–Cas9.82 RBCs were selected as the source of EVs because of their abundance, lack of genomic DNA, and demonstrated safety and scalability. Group O blood from healthy donors was used in this study to minimize immune response because of the lack of surface antigens.82,83 The EVs purified from RBCs were electroporated in order to incorporate the Cas9 mRNA and sgRNA targeting the locus of oncogenic miR-125b-2. The EVs were able to efficiently cleave the miR-125b locus in monocytic leukemia MOLM13 cells in culture.82

3.4 Immune concerns regarding Cas9 mRNA

Immune complications associated with Cas9 mRNA delivery come from not only the Cas9 mRNA itself, but also the sgRNA (if not encoded in a plasmid). In vitro synthesized sgRNA can activate PRR RIG-I and induce an innate immune response through type I interferon signaling if not modified correctly (inclusion of 5′-triphosphate group).84 In addition, because of its bacterial origin, Cas9 mRNA can activate TLR7 and TLR8 (Fig. 3).85–87 Fortunately, it has been shown on numerous occasions that chemical modification of the mRNA, in addition to preventing degradation, can allow for an ablation of the immune response.12,84–87 Despite the apparent drawbacks of using mRNA delivery of Cas9, techniques such as chemical modification and encapsulation via biomaterial vectors allow for a more robust experience, thus maintaining its exigence.

3.5 Common characteristics of materials used for Cas9 mRNA delivery

Similar to Cas9 plasmid, Cas9 mRNA carries a net negative charge. Therefore, many strategies are aimed at complexing the mRNA with positively charged groups to improve cell entry. The majority of vectors are also cationic liposomes, but other vectors including functionalized amino lipid nanoparticles and extracellular vesicles are also being used (Fig. 1b). One of the main advantages of using Cas9 mRNA is not having to access the nucleus since mRNAs work in the cytosol of cells. However, due to its intrinsic susceptibility to degradation and hydrolysis, proper modifications should be made to the mRNA and to the biomaterial vectors to improve stability in circulation. PEGylating the surface of nanoparticles is a commonly used strategy for materials modification (Fig. 2).
image file: c8bm01310a-f2.tif
Fig. 2 Examples of nanoparticle design strategies for improving Cas9 delivery. Incorporating polyethylene glycol (PEG) on the surface of nanoparticles can improve the stability and circulation by reducing the aggregation and non-specific protein binding. Zwitterions may provide another strategy for creating an anti-fouling surface, but more work must be done to verify its efficacy, specifically in Cas9 delivery systems.139,140 Surface modifications can be done to add components for enhancing cell entry. These components include cell penetrating peptides, positively charged functional groups, cationic lipid formulations and targeting ligands including RGD, hyaluronan, folate, etc.58,61,98 For endosomal escape, strategies such as the proton sponge effect and direct interaction with the endosomal membrane are often used. PEI, dendrimers, cell penetrating peptides, DOPE, zeolitic imidazolate frameworks, black phosphorus sheets and endosome-disruptive polymer PAsp(DET) have all been demonstrated to be effective for this purpose.12,33,34,37,38,40,48,59–61,65,66,72,77,80,94–98,104,105,107,108,111,112

4. Delivery of Cas9 in its protein form

Delivering Cas9 in its protein form allows for the most direct and quickest action. The Cas9 nuclease can directly form the ribonucleoprotein (RNP) complex with the sgRNA and traverse the nucleus for gene editing. It avoids some of the drawbacks of Cas9 mRNA and plasmid DNA such as susceptibility to degradation and integration risks. The protein or RNP method has also been shown to perform better in cells that are difficult to transfect.4,6,88 Because of these advantages and its immediate onset of action, Cas9 RNP offers great potential for translational applications and has been the most extensively investigated platform for studies attempting HDR. Furthermore, the potential for translational applications can be further maximized through the use of non-viral, biomaterial vectors as shown in the studies discussed in this section.

4.1 The issue with charge

A consideration to be made for the delivery of Cas9 protein is its positive charge unlike mRNA and plasmid DNA.89 While this allows for favorable interactions with the negatively charged cell membrane, its inability to directly complex with cationic lipid or polymer formulations would leave the protein susceptible to degradation by proteases.90 Two strategies are often used to circumvent this issue. One strategy is to modify the Cas9 protein with a negatively charged molecule (e.g. an anionic peptide or protein). A more common approach is to complex Cas9 protein (+22 net theoretical charge) with an sgRNA (∼100 anionic phosphate groups) to form negatively charged RNP. Zuris et al. investigated these two strategies for the delivery of Cas9 protein.91 In this study, Cas9 was fused with an engineered, highly negatively charged GFP molecule. This gave the complex an overall net negative charge, thus allowing for efficient encapsulation with the commercially available cationic lipid based formulation, RNAiMAX. In vitro results obtained using the fusion protein showed a higher editing efficiency in U2OS cells than when using Cas9 plasmid delivered with Lipofectamine 2000. It was found, however, that fusion with the negatively charged GFP molecule resulted in less efficient gene cleavage as compared to native Cas9 protein complexed directly with sgRNA. Multiplexed gene editing was also demonstrated using Cas9 RNP with different sgRNAs in the study. The Cas9 RNP was then delivered in vivo to mice cochlea using Lipofectamine 2000 as well as RNAiMAX, resulting in ∼20% target gene modification when delivered with Lipofectamine 2000. Despite the reduction in efficiency after fusing the negatively charged protein to Cas9, the study demonstrated a general delivery strategy for gene editing tools including Cre tyrosine recombinase and TALE transcription activators in addition to the Cas9 nuclease.91

4.2 Biologically-inspired delivery systems

The use of biologically inspired delivery systems has been shown by Sun et al. through a novel DNA nanoclew (DNA NCs) carrier particle.92 Yarn-like DNA nanoclews were synthesized through rolling circle amplification with palindromic sequences to drive self-assembly. Furthermore, DNA NCs were designed to partially bind to sgRNA, allowing for loading of the Cas9 RNP onto the NCs. PEI was then used to coat the DNA NCs to aid in endosomal escape. It was found that the design of the system itself played a crucial role in editing efficiency through the number of complementary pairs in the DNA NC structure binding to the sgRNA of the Cas9 RNP. DNA NCs were effectively delivered in vitro to U2OS osteosarcoma cells with minimal toxicity, and disrupted the target gene EGFP more efficiently compared to particles only containing Cas9 RNP and PEI. In vivo disruption of EGFP in U2OS tumors was also observed when DNA NCs were injected intratumorally. While DNA NCs were able to effectively deliver the CRISPR–Cas9 system, it were suggested that further studies needed to be done to assess their potential immunogenicity.92

4.3 Modifying the Cas9 protein for nanoparticle assembly

Modification of Cas9 protein can provide new delivery strategies by introducing additional interactions to various delivery platforms. In a recent study, one type of EVs, arrestin domain containing protein 1 [ARRDC1]-mediated microvesicles (ARMMs), were used to deliver the Cas9 RNP to U2OS osteosarcoma cells.93 The use of microvesicles in this study was considered advantageous to exosomes as controlled production was feasible through simple overexpression of ARRDC1. In addition, ARMMS are known to recruit endogenous proteins, thus suggesting that other macromolecules may also be packaged in a similar manner. The authors were able to package the Cas9 RNP by fusing WW domains to Cas9, which allowed it to interact with the PPXY motif on ARRDC1 and be incorporated into the ARMMs after transfection of HEK293T cells.93 In comparison, little Cas9 was found in ARMMs without the WW-domain fusion. Fusion did not affect the activity of Cas9, and ARMMs carrying the modified Cas9 RNP were able to significantly reduce the expression of the reporter gene GFP.93

As the overuse of antibiotics may lead to an emergence of drug-resistant bacteria, CRISPR–Cas9 offers a potential solution for counteracting this serious public health concern. Kang et al. were able to use the structural properties of the Cas9 protein itself to drive the assembly of nanoparticles. Branched PEI was reacted with sulfo-SMCC, which then acted as a cross-linker and reacted with free thiol groups found on the cysteine residues of Cas9.94 The cationic PEI-Cas9 was then complexed with sgRNA targeting mecA, the main gene responsible for methicillin resistance in methicillin-resistant Staphylococcus aureus (MRSA). The nanocomplex was shown to have greater bacterial uptake in comparison with a simple mixture of unmodified Cas9 RNP with PEI or Lipofectamine. This finding is significant as one of the greatest challenges in the delivery of therapeutics to Gram positive bacteria is the need to traverse the cell wall in addition to the cell membrane. Furthermore, MRSA treated with the nanocomplex and then subjected to antibiotic treatment was found to show reduced growth rates, decreased numbers of colony forming units, and reduced relative growth in comparison with Cas9 RNP delivered using Lipofectamine.94

4.4 The effects of lipid chemical structure on delivery efficiency

Wang, M. et al. generated a library of lipid nanoparticles with bioreducible disulfide bonds incorporated into the hydrophobic tail.95 This allowed for improved degradation and endosomal escape. Synthesis of the bioreducible lipids was done through a Michael addition of various amines and an acrylate featuring a disulfide bond and a 14-carbon hydrocarbon tail. Lipid nanoparticles consisting of cholesterol, DOPE, C16-PEG2000-ceramide, and bioreducible lipids with varying amine head groups were formulated for the delivery of Cas9 RNP. Using EGFP as a target gene in HEK cells, it was shown that the structure of the amine head group had a significant impact on endosomal escape and gene editing efficiency. The use of varied amine head groups highlights the relevance of structure–activity dynamics in nanoparticles used for CRISPR–Cas9 delivery.95

4.5 Supramolecular assembly for creating delivery nanoparticles

As covalent fusion of cell-penetrating peptides or other delivery components to Cas9 protein may reduce the endonuclease's activity, a non-covalent strategy such as supramolecular assembly or complexation can be employed. Supramolecular interactions as shown by Lostalé-Seijo et al. allowed for the design of amphiphilic peptide vectors with high delivery efficiency of non-modified Cas9.96 A pro-helical cationic peptide with hydrazide moieties was reacted with hydrophobic aldehydes to generate an amphiphilic structure. This amphiphilic penetrating peptide was then incubated with Cas9 RNP to form nanoparticles through supramolecular complexation, resulting in delivery vehicles capable of crossing the cell membrane and disrupting endosomes. Compared to Lipofectamine 2000, the amphiphilic nanoparticles exhibited lower cytotoxicity and greater indel formation in the EGFP gene at lower doses of Cas9 RNP in HeLa cells. In another study, Endo-Porter, an amphiphilic α-helical peptide, was designed for the delivery of Cas9 RNP.97 It is hypothesized that the weak-base histidine residues of EP can facilitate endosomal escape by the proton sponge effect. The peptide and Cas9 RNP form nano-size complexes via electrostatic interactions. These complexes were able to generate indels at a frequency of 40.4% in the GFP transgene in GFP-J774A.1 macrophage cells, and greater GFP loss was observed in comparison with Lipofectamine RNAiMAX. The peptide-Cas9 RNP complexes were also tested for in vivo gene editing. Indels of GFP transgene were observed in 3% of peritoneal exudate cells isolated from mice after 5 days of daily intraperitoneal injections. Finally, the complexes with sgRNA targeting the gene Nrip1, a potential therapeutic target for type 2 diabetes and other metabolic disorders, were delivered to primary white pre-adipocytes, and an indel frequency of up to 43.8% in the Nrip1 gene was achieved.

Host–guest interaction provides a well-tolerated method for generating reversibly crosslinked nanoparticles that show high Cas9 loading efficiency.98 Chen et al. generated a PEI hydrogel core formed through the interactions between cyclodextrin (host)-engrafted branched PEI and adamantane (guest)-engrafted branched PEI. The core was encapsulated by a DOTAP liposome and conjugated with a cell penetrating peptide, mHph3. The encapsulation efficiency of Cas9 protein in these nanoparticles was substantially higher than that of conventional DOTAP liposomes (62.8% vs. 6.3%), which was likely due to Cas9's interaction with the PEI hydrogel core. In addition, the use of guest–host interactions allowed for the formation of PEI hydrogel nanoparticles without using highly reactive initiators that can interfere with protein activity. DNA minicircles were then used to provide sgRNA targeting PLK-1 as minicircles have been shown to enhance transgene expression in comparison with regular plasmids.99 The liposome–hydrogel nanoparticles were able to successfully deliver Cas9 and sgRNA minicircles to U87 and GS5 cells in vitro and reduce cell proliferation due to the inhibition of the PLK-1 gene. In order to enhance tropism to tumors, a tumor penetrating peptide, iRGD, and Lexiscan, a drug that is known to transiently increase blood–brain barrier permeability, were incorporated into the nanoparticles. The iRGD and Lexiscan modified nanoparticles showed enhanced tumor accumulation in an intracranial U87 tumor model following intravenous injection. Furthermore, reduced tumor PLK-1 expression and increased survival were observed in mice treated with these nanoparticles.98

4.6 The use of inorganic materials for Cas9 RNP delivery

Besides organic materials, inorganic nanoparticles have also been used for the delivery of Cas9 because of their distinctive chemical and physical properties. For example, gold nanoparticles have shown good chemical stability, structural tunability and biocompatibility as drug carriers. In addition, they have unique optical and plasmonic properties and can be easily modified with surface ligands or proteins through gold–thiol reaction.100 Recently, Mout et al. developed a versatile, cationic gold nanoparticle platform with an arginine coating (ArgNPs) for improved membrane transport.101,102 Engineered Cas9 carrying a glutamate peptide tag and NLS was used as aglutamic acid provided negative charge sites for the Cas9 RNP to bind to the cationic ArgNPs. Interestingly, it was observed that the cellular internalization of Cas9-ArgNPs was through a cholesterol-dependent membrane-fusion-like process but not via endocytosis. In addition, the cytosol delivery was associated with the length of the glutamate peptide tag. As a demonstration of gene editing, Cas9-ArgNPs targeting the PTEN or AAVS1 genes led to ∼30% indel in these genes in HeLa cells. The versatility of this delivery system was also demonstrated in another study where Sirp-α knockout RAW264.7 macrophages were generated using Cas9-ArgNPs to enhance their phagocytosis of cancer cells.103 Using the same AuNP system as that discussed previously for Cas9 plasmid delivery,65 Wang, P. et al. showed that the delivery system could also be used to deliver Cas9 protein,104 which further demonstrated the versatility of gold nanoparticle-based delivery systems.

Effective delivery of the CRISPR–Cas9 system to various cell types in the brain that are known to be hard to transfect will open up the possibility to treat various neurological disorders. A recent study used another gold nanoparticle-based system, the CRISPR-Gold platform, to achieve intracranial delivery of Cas9 RNP and edit all of the major cell types of the brain.105 The CRISPR-Gold system consisted of gold nanoparticles modified with ssDNA, and Cas9 RNP was complexed to the particles. A thin layer of silica (to increase the negative charge density) and an endosomal disruptive polymer poly(N-(N-(2-aminoethyl)-2-aminoethyl) aspartamide) (PAsp(DET)) were subsequently coated on the particles to form the CRISPR-Gold system. The CRISPR-Gold showed little adverse effect on cultured primary neurons. To demonstrate genome editing in an adult mouse brain, CRISPR-Gold was delivered to the hippocampus and striatum of Ai9 mice for deletion of the stop codon for tdTomato expression. Approximately 10–15% of cells in the injected area were tdTomato positive, suggesting that specific DNA sequences could be targeted in the brain using CRISPR-Gold. Furthermore, it was shown through cells expressing tdTomato that astrocytes, microglia and neurons could all be edited. The Fmr1 knockout mice strain was then used as a model for fragile X syndrome (FXS) and the gene Grm5 was chosen as the target due to studies demonstrating the involvement of mGluR5 signaling in FXS. Intracranial delivery of CRISPR-Gold to the striatum of these mice resulted in 14.6% indel formation in the Grm5 gene.105

Ultrathin 2D nanomaterials such as graphene oxide (GO) are rising in popularity as delivery vehicles due to properties such as a large surface-to-volume ratio, biocompatibility and stability.106 A recent study demonstrated that GO could also be used to deliver Cas9 RNP.107 In this work, GO nanoparticles were functionalized with PEG and PEI to enhance the colloidal stability and endosomal escape, respectively. Cas9 RNP was then immobilized onto the surface of the GO-PEG-PEI particles via physical adsorption and π-stacking interactions. It was hypothesized that the immobilization of Cas9 RNP on the GO-PEG-PEI surface could protect the Cas9 RNP from enzymatic degradation. This hypothesis was confirmed by showing that GO-PEG-PEI:Cas9 RNP could function in the presence of RNase. GO-PEG-PEI:Cas9 RNP nanoparticles targeting EGFP or CXCR-4 model genes showed ∼30–50% gene expression inhibition in AGS cells. In addition, low cytotoxicity was observed when the AGS cells were transfected with the nanoparticles.

Black phosphorus (BP) nanosheets are another class of ultrathin 2D materials growing in popularity as drug carriers due to their biodegradability and high surface-to-volume ratio.108 Zhou et al. used BP nanosheets as a platform for loading the Cas9 RNP via electrostatic interactions and creating a biodegradable delivery vehicle.109 The Cas9 RNP was modified with 3 NLS peptides that served dual purposes of increasing electrostatic interactions with the BP nanosheets and improving nuclear transport. A high loading capacity of 98.7% was observed. The Cas9 RNP showed effective endosomal escape and nuclear transport when delivered using BP nanosheets. It was proposed that the accelerated degradation of BP in acidic endosomes released a large quantity of phosphite/phosphate ions, which elevated osmotic pressure and induced endosome bursts. Gene editing using BP-Cas9 RNP nanosheets was subsequently demonstrated in multiple cell lines, achieving ∼20–30% indel frequency in the target genes. In addition, BP-Cas9 RNP nanosheets exhibited a higher gene editing efficiency than Lipofectamine CRISPRMAX in RAW 264.7 macrophages. Intratumoral administration of BP-Cas9 RNP targeting EGFP also resulted in significant reduction of the EGFP signal in comparison with free Cas9 RNP in a mouse model.

Zeolitic imidazolate frameworks (ZIFs) are a class of metal organic frameworks containing imidazolate (C3H3N2) linkers and metal ions. They are pH sensitive, exhibit low cytotoxicity, and have highly porous structures that can encapsulate a variety of molecules.110 A recent study showed that these properties of ZIFs could be utilized to help in the delivery of Cas9 RNP. Nanoscale ZIFs containing Cas9 RNP were formed through the addition of 2-methylimidazole to a PBS solution of Cas9 and sgRNA. Afterwards, zinc nitrate was added, thus forming ZIF-encapsulated Cas9 RNP. The ZIF-Cas9 RNP nanoparticles delivered to CHO cells showed enhanced endosomal escape and greater reduction of target gene expression as compared to Lipofectamine. It was proposed that the pH buffering capacity of the imidazole linkers aided in endosomal release through protonation of the imidazole ring and subsequent release of the Cas9 RNP.111

4.7 Homology directed repair using Cas9 RNP

Pioneering work was conducted by Lee et al. using homology directed repair to correct the mutation for Duchenne muscular dystrophy. The previously discussed CRISPR-Gold delivery system was used in this study with the ssDNA coating being replaced with the donor template.112 Using gold as the core material allowed for a densely packed layer of donor DNA to be easily coated onto the surface. CRISPR-Gold induced HDR was first studied in vitro using human embryonic stem cells, human induced pluripotent stem cells, dendritic cells, and primary myoblasts from mdx mice. It was found that the HDR efficiency for all cell types was between 3 and 4% (targeting the CXCR4 or the dystrophin gene), and it was significantly more effective than when using Lipofectamine or nucleofection. Intramuscular administration of CRISPR-Gold targeting mutated dystrophin gene under therapeutically relevant conditions showed 0.8% HDR efficiency, which was enough to enhance strength and agility of the mice. In addition, deep sequencing analysis revealed minimal off-target DNA damage as well as no noticeable elevation of inflammatory cytokines in comparison with PBS.112 This is one of the first studies to demonstrate impressive HDR results using a non-viral vector.

One of the key challenges to HDR is the incorporation of the donor template to the Cas9 RNP. The recently developed S1mplex system provides an efficient method to preassemble the Cas9 RNP with a donor nucleic acid prior to delivery. This would reduce the time associated with gene editing applications, and potentially increase the frequency of precise gene editing. The system consists of Cas9 protein, sgRNA with a streptavidin-binding S1m aptamer, streptavidin, and a biotinylated single-stranded oligodeoxynucleotide donor template.113 In an effort to optimize the delivery of the S1mplex system, Wang, Y. et al. developed a polyplex vector composed primarily of the cationic poly(N,N′-bis(acryloyl)cystamine-co-triethylenetetramine) polymer with additional cross-linking capabilities for increased stability.114 The cationic polymer included imidazole groups and disulfide bonds, thus allowing for encapsulation, cellular uptake, endosomal escape and cytosolic unpacking capabilities in one system. PEG was also included in the polyplex to shield the positive charge, enhance stability and prevent non-specific adhesion. The polyplexes containing Cas9 RNP were delivered to mCherry-expressing HEK 293 cells and demonstrated similar transfection efficiencies to Lipofectamine 2000. For the assessment of HDR, the polyplexes containing S1mplex cargo that was designed to modify the BFP transgene to GFP were delivered to BFP-expressing HEK 293 cells. The efficiency at editing the BFP gene to GFP was assessed using flow cytometry and an editing efficiency comparable to Lipofectamine 2000 was observed. However, the polyplexes showed significantly lower cytotoxicity in comparison with Lipofectamine 2000.

4.8 Immune concerns regarding Cas9 proteins

As recombinant proteins such as Cas9 are often expressed in bacterial hosts, they must be highly purified so that bacteria-derived lipopolysaccharide (LPS) endotoxins do not trigger innate immune responses by activating LPS-binding protein, CD14, and TLR 4 (Fig. 3).115 As purification levels as high as 95% are still sufficient for inducing an immune response, alternative methods for expressing the recombinant protein can also be investigated to avoid endotoxin contamination, especially for applications involving cells that are sensitive to endotoxins.116
image file: c8bm01310a-f3.tif
Fig. 3 The immune challenges to CRISPR/Cas9 delivery. Nanoparticles carrying the CRISPR/Cas9 system face numerous challenges from both the innate and adaptive immune responses. In circulation, innate immune responses such as clearance by macrophages and other phagocytes can occur for nanoparticles. Proper modifications should be made to the Cas9 component so that activation of toll-like receptors (TLRs) or other pattern recognition receptors (PRRs) does not occur in the target cell. For example, the mRNA form of Cas9 can activate TLR7 and TLR8 because of its bacterial origin, and CpG motifs on plasmids derived from bacterial hosts can activate TLR9. Furthermore, impurities such as endotoxins in plasmid and protein samples can be detected by TLR4. Activation of the TLRs will result in the expression of pro-inflammatory genes. Plasmids have also been shown to activate the cGAS-STING pathway, leading to the expression of pro-inflammatory genes. These intracellular responses highlight the importance of modifying the Cas9 cargo to reduce the immune response and maintain efficacy. The adaptive immune response may be activated through several routes including a pre-existing immunity to the Cas9 protein. While PEGylation is a common surface modification used to reduce phagocyte clearance and opsonization, it has been shown to induce an adaptive immune response as shown through reports detecting the presence of antibodies reactive to PEG. Upon entry into cells, endosomal escape can result in MHC class I antigen processing and presentation while retention in the endosome leads to MHC class II antigen processing and presentation, which may then elicit cellular and humoral immune responses depending on the presence of other immunostimulatory signals.

Because the most common forms of the Cas9 protein are derived from the bacteria Staphylococcus aureus (S. aureus, saCas9) and Streptococcus pyogenes (S. pyogenes, spCas9), it is intuitive to consider a possible immune response to Cas9 proteins. Indeed, Charlesworth et al. discovered pre-existing adaptive immune responses specific to Cas9. Serum IgG antibodies against spCas9 and saCas9 were detected in 60–80% of donors.25 The overall findings are supported in a study by Simhadri et al., who detected the presence of anti-SpCas9 and anti-SaCas9 antibodies in various human sera at a frequency of 2.5% and 10% respectively, although the assay format and the number of samples evaluated differed between the two studies.26 While anti-spCas9 T cells were not detected, anti-saCas9 T-cells were found in 46% of donors.25 These pre-existing adaptive immune responses to Cas9 will likely add complications when using CRISPR/Cas9 for clinical applications.

Adaptive immune responses against Cas9 can also be elicited during in vivo application of the gene editing tool. Wang et al. discovered significant IgG1 antibody response specific to spCas9 in mice that were treated with adenovirus vector carrying spCas9.21 These results were replicated in other mice strains, and additional SpCas9-specific IgG2a and IgG2b antibodies were discovered. Culturing primary splenocytes from spCas9:sgRNA treated mice revealed an increase in IL-2 secretion distinct from the control conditions, suggesting the possible development of CD4 and/or CD8 T-cell responses.21

Despite the still limited number of reports on immune reactivity against the Cas9 protein, future studies should continue to investigate the possibility of innate and adaptive immune responses in order to assess their potential effects on the applications of CRISPR–Cas9. The tragic accident from a previous gene therapy trial should serve as an important lesson about the significance of immune responses to gene therapies.67 One strategy that may circumvent adaptive immune responses against Cas9 is to deliver them using biomaterials that lack adjuvant effects or are less immunostimulatory.117

4.9 Common characteristics of materials used for Cas9 protein delivery

Cas9 in the protein form provides the quickest onset of action, but it must still reach the nucleus in order to achieve its therapeutic effect. Even though the Cas9 RNP is often provided with NLS for traversing the nuclear membrane, protection along its route to the nucleus could help to increase the likelihood of successful gene editing. Currently, the most commonly employed vectors include liposomes, Cas9/cationic peptide complexes and gold nanoparticles (Fig. 1b). These vectors allow for successful cell entry and promote endosomal escape of the Cas9 RNP. Direct chemical or physical modifications of Cas9 RNP that are difficult to achieve through the plasmid or mRNA route may provide additional advantages when using the protein form in the future.

5. Conclusion

This review provides a comprehensive resource for the delivery of the CRISPR–Cas9 system using biomaterial-based vectors (Table 1). The numerous types of delivery systems and the multifaceted functions of their components (Table 2) have demonstrated the versatility and tunability of biomaterials, making them an attractive option for addressing many of the biological problems associated with the delivery of CRISPR–Cas9.

However, despite their numerous advantages, biomaterial vectors have many issues of their own. While synthetic materials possess the tunability desired for creating tailored nanoparticles, they typically lack bioactive motifs for influencing many of the cellular processes. Naturally derived biomaterials possess high biocompatibility and bioactivity, but often have issues related to batch-to-batch variability and difficulty in processing. Despite these drawbacks, new materials delivery systems that exhibit tunable physical properties along with biocompatibility are increasingly being developed because of the advances in chemistry and materials science. The immune-related complications that surround biomaterial CRISPR–Cas9 systems are also an important issue. They are two-fold: one stems from immune responses associated with the payload, while the other is immunogenicity of the biomaterials themselves (Fig. 3). For example, PEG is commonly employed on the surface of nanoparticles to reduce protein adsorption, increase blood circulation time and offer protection against certain innate immune response.54,118 However, studies have shown that the benefits of PEGylation are reduced due to the possible presence of antibodies reactive to PEG, though more work is needed to refine anti-PEG assays and develop reference sera.119–122 Certain lipids have also been shown to elicit innate immune response and/or adaptive immune responses.123–126 It is important to consider these effects in order to maximize the therapeutic potential of CRISPR–Cas9 delivery and gene editing.

With the emergence of techniques to generate new biomaterials and an increased understanding of the biological challenges associated with extracellular and intracellular delivery of various forms of CRISPR/Cas9, it is expected that tailored biomaterial vectors with high efficiency, high tissue/cell specificity, low immunogenicity, and good temporal control will be developed and will greatly accelerate the clinical translation of CRISPR/Cas9.

With great power comes great responsibility. The recent announcement about the first genome-edited babies has shocked the world.127 On the one hand, it exposed the potential impact CRISPR–Cas9 and other gene editing platforms could have in our lives. But on the other hand, more importantly, in order to apply such technologies, we need to continue investigating their long-term effects and discussing the ethical and moral implications of their use.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We would like to thank Angela Park for her help with the illustrations and Joo Ho Kim, Yi Zuo, Zhiwei Fang, and Supeng Ding for helpful and thoughtful discussions.

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