Discover the recent progress of nonviral delivery carriers for CRISPR/Cas9 systems

The CRISPR/Cas9 system has recently been recognized as a versatile and robust genome-editing platform for constructing disease models and rectifying genes affected by disease.

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CRISPR, an acronym denoting clustered regularly interspaced short palindromic repeat, was identified by Nakata et al. in /US/en1987/US/en1 as repeat sequences interspaced by nucleotide spacers in the Escherichia coli genome.

This genome-editing system comprises two primary components: a nuclease protein Cas9, binding to DNA and initiating double-strand breaks (DSBs), and a very short single guide RNA (sgRNA) directing the Cas9 nuclease to the targeted genomic locus.

As the CRISPR/Cas9 system exclusively functions within the cellular environment, a delivery mechanism to target cells is necessary.

Physical approaches and viral vectors have both been extensively studied for CRISPR/Cas9 delivery, demonstrating significant progress in improving delivery accuracy and transfection efficiency.

Nevertheless, these approaches possess inherent limitations, encompassing insertional mutagenesis, carcinogenesis, and immunogenicity, which markedly confine their clinical applications. Hence, there is a need for novel CRISPR/Cas9 delivery systems to facilitate the secure and efficient clinical advancement and utilization of CRISPR/Cas9.

Effective in vivo delivery of the CRISPR/Cas9 system requires comprehending and addressing the three major challenges that span from in vitro loading to in vivo delivery of CRISPR/Cas9.

First and foremost, the size of the CRISPR/Cas9 system is much greater than that of conventional gene editing tools. Specifically, the overall plasmid size exceeds 7 kb, and commonly used Cas9 proteins are approximately /US/en160 kDa, significantly heightening the difficulty of in vivo delivery.

Consequently, it is vital to condense the CRISPR/Cas9 system into comparatively small particles through the utilization of suitable carriers.

Secondly, the uncovered CRISPR/Cas9 genome editing cargos consist of exogenous bio-macromolecules that are susceptible to recognition and swift clearance by the host immune system.

Thus, their half-life is too short for effective in vivo functionality. To extend the half-life, the delivery platform must shield the genome-editing cargo from degradation in the physiological environment.

Thirdly, the gene editing process unfolds in the cytoplasm or nucleus, yet genome-editing cargos encounter difficulty independently traversing the hydrophobic lipid cell membrane. Hence, a suitable delivery vector becomes imperative to facilitate the penetration of the CRISPR/Cas9 system into host cells.

In earlier investigations, non-viral nanoparticles such as liposomes, polymeric nanoparticles, and inorganic nanoparticles have showcased numerous advantages, including lower immunogenicity, high packaging capability, and design flexibility. These attributes indicate significant potential in meeting the demands for CRISPR/Cas9 delivery.

Recent research endeavors have concentrated on non-viral methods as promising alternatives for crafting an efficient CRISPR/Cas9 delivery system. This article will provide a summary of the latest non-viral approaches employed for CRISPR/Cas9 delivery, serving as a reference for future research.

Lipid-based platforms

Lipid-based carriers represent a prominent category of non-viral gene carriers. Lipofectamine, a commercially available liposome, has been extensively employed in vitro for mRNA transfection due to its notable transfection efficiency.

However, its widespread use has been curtailed by high toxicity and inflammatory side effects, limiting further application.

Researchers are, therefore, actively seeking ways to modify conventional liposomes by incorporating biodegradable chemical bonds or targeting ligands to enhance functionality.

Wang et al.,² for instance, presented a combinatorial library of cationic bioreducible lipids for delivering Cas9/sgRNA. The lipid-based vectors effectively enabled the escape of Cas9/sgRNA from endosomes, directing the protein to its intracellular targets under a reductive intracellular environment, achieving over 70% delivery efficiency.

Finn et al.³ designed a biodegradable lipid-based formulation utilizing labile ester linkages. Introducing apolipoprotein E (ApoE) as the target moiety significantly enhanced uptake by hepatocyte cells, resulting in more than 97% gene knockdown efficiency.

Exosomes, small extracellular vesicles secreted by mammalian cells, are attractive nanocarriers due to their stability, biocompatibility, low immunogenicity, and low toxicity. However, encapsulating large nucleic acids or proteins into exosomes is challenging due to their small size (30–100 nm).

To leverage exosomes' advantages in CRISPR/Cas9 delivery, Lin et al.⁴ developed a hybrid exosome by co-incubating them with liposomes. Unlike the original exosomes, the hybrid encapsulated CRISPR/Cas9 plasmid DNA and efficiently expressed the encapsulated genes in mesenchymal stem cells (MSCs).

Polymer-based platforms

Polymer-based nanoparticles have gained widespread use in delivering different types of nucleic acids, such as plasmid DNA, RNA, and oligonucleotides. Their popularity stems from their superior encapsulation ability, the potential for targeted delivery to specific tissues or organs, and their effectiveness in stabilizing nucleic acids against serum-induced aggregation.

Reflecting on past research, the use of polymer-based platforms for CRISPR/Cas9 delivery has become a significant area of interest.

Polyethyleneimine (PEI) is the most prevalent polymeric gene vector. The abundant amine groups in PEI provide it with high charge density, facilitating efficient DNA condensation and endo/lysosomal escaping.

Presently, PEI 25K is even deemed the gold standard for DNA/RNA transfection. However, the excessively strong positive charge and considerable cytotoxicity impede further in vivo applications.

Encouragingly, recent studies indicate that this cytotoxicity can be effectively reduced by employing linear and lower molecular weight PEI or through chemical modification with moieties such as PEG.

Wei and colleagues⁵ presented a multifunctional nucleus-targeting "core-shell" artificial virus (RRPHC) designed for the delivery of the CRISPR/Cas9 system. This RRPHC artificial virus features a core-shell structure. The core is composed of fluorinated low molecular weight PEI, while the shell is formed using a versatile multifunctional PEG layer, specifically RGD-R8-PEG-HA, RRPH.

This artificial virus efficiently facilitates the endosomal escape and nucleus entry of the CRISPR/Cas9 system, eliminating the need for any additional nuclear localization signals. It demonstrates exceptionally high transfection efficiency in SKOV3 cells, achieving rates exceeding 90%.

Likewise, Liu et al.⁶ devised a multistage delivery nanoparticle (MDNP), incorporating phenylboronic acid (PBA)-modified low molecular weight PEI and 2,3-dimethylmaleic anhydride (DMMA)-modified mPEG113-b-PLys100.

The distinct surface properties at various delivery stages ensure the efficient delivery of the CRISPR/dCas9-miR-524 system by this multistage delivery nanoparticle.

In a separate study, Zhang and colleagues⁷ synthesized polyethyleneimine-β-cyclodextrin (PC) as a carrier for delivering Cas9/sgRNA pDNA in vitro. Due to its structural similarity to high molecular weight PEI, PC serves as an effective delivery vector with high efficiency.

All three studies rely on low molecular weight PEI with functional chemical modification. In comparison to high molecular weight PEI, they exhibit similar transfection efficiency but lower cytotoxicity, underscoring their substantial potential as a safer alternative to PEI 25K.

Over the past decade, novel nanostructures crafted from DNA, such as DNA tetrahedrons, DNA origami structures, DNA nanorobots, and DNA nanoclews, have demonstrated significant potential in delivering the CRISPR/Cas9 system. This is attributed to their uniform size, biodegradability, and spatial addressability.

In 2015, Sun et al.⁸ introduced a synthetic DNA nanoclew (NC)-based carrier for the delivery of Cas9/sgRNA complexes both in vitro and in vivo.

With the aid of PEI and nuclear-localization-signal peptides, DNA NCs have displayed effective endosome escape and nucleus targeting capabilities, achieving notably higher editing efficacy (36%) compared to the cell-penetrating peptide (CPP)-based vector (9.7%).

Inorganic nanoparticles

In recent years, inflexible nanocarriers like carbon, gold, and other nanoscale inorganic materials have demonstrated efficacy in various gene delivery applications due to their high surface-to-volume ratio, size control, and colloidal stability in a physiological environment.

For instance, a nanocarrier comprised of cationic arginine gold nanoparticles (ArgNPs) and engineered Cas9 proteins has shown promise.⁹ This vector efficiently delivers protein and nucleic acid to the cytoplasm, subsequently transporting it to the nucleus, achieving an impressive 90% delivery efficiency.

In 2018, Alsaiari et al.¹⁰ introduced the groundbreaking use of nanoscale metal-organic frameworks (MOFs) for CRISPR/Cas9 RNP complex delivery. Nanoscale zeolitic imidazolate framework-8 (ZIF-8) effectively co-encapsulated both Cas9 protein and the negatively charged sgRNA with a high loading efficiency of 17%, thanks to its adjustable pore size.

With rapid endosomal escape and enhanced nucleus delivery, the CRISPR/Cas9 system encapsulated by ZIF-8 achieved a 37% reduction in the gene expression of green fluorescent protein over a period of four days.

In another study, Zhou et al.¹¹ utilized two-dimensional, biodegradable black phosphorus nanosheets (BPs) for CRISPR/Cas9 delivery. Cas9 RNP was loaded onto the BPs via electrostatic interaction with an impressive loading capacity of 98.7%.

The constructed delivery platform could enter cells through both direct membrane penetration and the endocytosis pathway, followed by effective endosomal escape upon biodegradation of the ultra-thin BPs. The resulting good biocompatibility and biodegradability make it a promising avenue for further research.

Hybrid materials-based platforms

In the realm of CRISPR/Cas9 delivery, no single carrier can comprehensively address all challenges. Combining the strengths of different materials into a hybrid multifunctional vector offers the capability to simultaneously meet various requirements, ultimately achieving more efficient delivery.

Polymeric nanoparticles coated with a SiO₂ shell have been proposed as promising hybrid carriers for safely and efficiently delivering biologically active compounds.

Timin et al.¹² devised a nanoparticle-based system by applying the layer-by-layer (LbL) self-assembly method to deposit poly-L-arginine hydrochloride and dextran sulfate on CaCO₃ particles.

After removing the CaCO₃ core and coating it with a SiO₂ shell, these carriers exhibited more efficient transfection than a commercially available liposome-based transfection reagent due to their high loading capacity and biocompatibility.

Liang et al.¹³ developed a PEG-PEI-Cholesterol (PPC) lipopolymer for delivering CRISPR/Cas9 plasmids encoding VEGFA gRNA. These aptamer-functionalized PPC lipopolymers successfully reduced VEGFA expression or secretion, demonstrating significant inhibition in orthotopic osteosarcoma malignancy and lung metastasis.

In another study, Chen et al.¹⁴ created liposome-templated hydrogel nanoparticles (LHNPs) for targeted CRISPR/Cas9 delivery.

The PEI-based hydrogel features a core-shell structure, wherein the core consists of a cationic polyplex formed by crosslinking cyclodextrin (CD)-engrafted PEI (PEI-CD) with adamantine (AD)-engrafted PEI (PEI-AD), while the shell is composed of DOTAP lipids.

This distinctive core-shell structure can deliver Cas9 protein and plasmid DNA simultaneously, showcasing high delivery efficiency and low toxicity.

Graphene oxide (GO) has also garnered attention in biological applications, particularly as a carrier for delivering small molecular drugs and nucleic acids into cells. The planar structure of GO provides a higher specific surface area compared to other nanocarriers, effectively enhancing payload capacity.

In 2018, Yue et al.¹⁵ pioneered the development of the first delivery platform based on PEG and PEI dual-functionalized graphene oxide (GO), capable of loading Cas9/sgRNA through physical adsorption and π-stacking interactions.

This nanoplatform successfully achieved intracellular delivery of Cas9/sgRNA via endocytosis, resulting in 39% gene disruption with excellent biocompatibility.

Research has indicated that hybrid multifunctional vectors possess increased intelligence, allowing them to simultaneously overcome intracellular and extracellular barriers.

For instance, Harashima et al.¹⁶ introduced a multifunctional envelope-type nanodevice (MEND) composed of a mitochondria-targeting liposome called MITOPorter and enzymatically cleavable PEG.

Such intelligent nanocarriers have the potential to evade recognition by the host immune system during blood circulation and achieve effective cell internalization after accumulating in tumor tissues, ultimately achieving high delivery efficiency.

Wang et al.¹⁷ devised a photothermal-activatable lipid/gold nanoparticle (AuNP) platform for delivering Cas9-gPlk-1 plasmids. This multifunctional vehicle enters tumor cells and releases the Cas9-gPlk-1 plasmids into the cytosol through laser-triggered thermo-effects of the AuNPs.

Guided by the TAT peptide, the Cas9-gPlk-1 plasmids then enter the nuclei to complete the gene editing process. The synergistic effects of AuNPs, TAT peptide, and the outer lipid shell ensure high efficiency and targeted gene editing.

Conclusion and perspectives

The CRISPR/Cas9 system presents notable advantages over other genome-editing technologies, such as simplicity and versatility. However, its clinical application heavily relies on the efficient delivery of genome-editing cargo to target cells.

Numerous innovative nanoparticle delivery systems, including polymer-based, lipid-based, and rigid inorganic nanoparticles, have been designed for the CRISPR/Cas9 system.

In response to the diverse requirements at different in vivo delivery stages, hybrid multifunctional delivery platforms are also under development. All these advancements make the clinical application of non-viral vectors for CRISPR/Cas9 delivery very promising in the near future.

To date, most delivery platform designs are tailored for plasmid DNA (pDNA) or mRNA. In comparison, direct delivery of Cas9 protein/sgRNA offers advantages such as rapid action, high efficiency, and lower off-target effects.

However, encapsulating the ribonucleoprotein (RNP) into a small particle while maintaining its biological activity in the bloodstream remains a significant challenge. There are limited alternative protein carriers for delivery, so exploring this avenue is a crucial and much-needed research direction to advance CRISPR/Cas9 technology.

Acknowledgments

This research received support from the National Key Research and Development Programs of China (2018YFA0209700), the National Natural Science Foundation of China (NSFC; Grant Nos. 51673100), and the Fundamental Research Funds for the Central Universities at Nankai University (Grant Nos. ZB19100123).

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