CRISPR Delivery Methods: Cargo, Vehicles, and Challenges

CRISPR viral delivery can be achieved through the use of adeno-associated viral vectors (AAVVs), full-size adenoviral vectors (AdVs), and lentiviral vectors (LVs). Viral vectors are particularly popular for in vivo CRISPR experiments and clinical trials, however, they also lend themselves to ex vivo and in vitro studies, and are useful in studies requiring long-term expression of CRISPR components.

Researchers have also more recently developed virus-like particles (VLPs), which have some of the advantages of viral delivery vehicles without some of their disadvantages. HEK293T cells are typically used to create viral particles containing the CRISPR cargo for all three of the CRISPR viral delivery methods described here. The viral particles are then used to infect the target cells, resulting in the continued production of CRISPR cargo within the host.

AAVs are small (~20nm), non-pathogenic viruses. Because they are not known to cause disease and have such mild immune responses in humans, they quickly became one of the most popular CRISPR delivery methods, specifically in the development of cell and gene therapies.

• Advantages: AAV CRISPR delivery has two key advantages: the adeno-associated virus is not inserted into the host cell genome, and host immune responses are typically milder than when other viral delivery methods are used. For these reasons, AAVs have received FDA approval for the treatment of certain diseases, making them the most common method used for preclinical models of disease as well as for in vivo CRISPR delivery.
• Disadvantages: The use of AAVs is often limited by the size of cargo that can be packaged within them; their 4.7kb payload capacity makes them too small to carry the Cas nuclease, sgRNA, and in the case of gene correction or insertion, a donor template for repair. Several strategies have been developed to overcome this hurdle. The first is to use AAVs to deliver only the sgRNA into cells that have been previously edited to express the Cas nuclease. The second is to package the sgRNA and Cas nuclease into separate AAVs with unique tags, co-transfect them into cells, then screen cells for successful co-transfection.

Another strategy to circumvent the payload restriction of AAVs is to use different CRISPR systems for editing. Several smaller, naturally occurring Cas variants have been discovered, while some nucleases have been engineered to minimize their size and achieve higher editing efficiencies. For example, the traditionally-used SpCas9 is 1368 amino acids in length, while Synthego’s high-fidelity hfCas12Max nuclease is only 1080aa.

AdVs are ubiquitous, with many serotypes present in the human population, causing diseases with varying degrees of severity. They can be used for CRISPR delivery, however, they are less suited to clinical applications than AAVs.

• Advantages: Similar to AAVVs, AdVs are not integrated into the host genome. Unlike AAVVs, however, they are not limited by size and can therefore carry much larger quantities of CRISPR cargo of up to 36kb. They can infect both dividing and non-dividing cells, can be grown in large quantities, and can be either replication-defective (RD) or replication-competent (RC). They can be modified to target specific cell types and to avoid immune responses.
• Disadvantages: AdVs can potentially cause undesirable immune responses and off-target effects. They can also result in damage to the host if they are inserted near key cellular proteins.

Lentiviruses are retroviruses causing many serious diseases in animals and humans. LVs have a backbone which is a provirus of HIV, and they are commonly used for CRISPR delivery in in vitro studies and in animal models.

• Advantages: LVs can be pseudotyped with envelopes from naturally occurring or engineered viruses, meaning that their infectivity of specific cell lines can be altered to suit the target. Like AdVs, they are able to deliver any size of CRISPR cargo. They can also infect both dividing and non-dividing cells.
• Disadvantages: Unlike AAVVs and AdVs, LVs are inserted into the host genome. The presence of an HIV backbone means the chance of integration into the host genome cannot be completely avoided and this has associated safety implications.

In an effort to overcome potential safety concerns associated with traditional viral vectors, researchers have also explored the use of virus-like particles (VLPs) for CRISPR delivery. VLPs are engineered particles; essentially an empty viral capsid, they contain no viral genome, making them non-replicative and non-integrating. They have been used to deliver a range of CRISPR components, including base editors and prime editors.

• Advantages: VLPs have the potential for cell and tissue-specific CRISPR delivery, and are also able to escape endosomes. Unlike real viral vectors, VLPs do not carry the associated safety concerns, such as integration. Additionally, they deliver CRISPR components in a transient fashion, which reduces the possibility of long-term expression and, therefore, off-target editing.
• Disadvantages: The clinical translation of VLPs for CRISPR delivery has been hindered by manufacturing challenges, and further research is needed to streamline and optimize their production at scale. There are also some issues with the stability of VLPs, as well as their cargo size limitations.

Non-viral methods for CRISPR-Cas9 delivery (nano delivery) include lipid nanoparticles, extracellular vesicles, lipoplexes and polyplexes, inorganic nanoparticles, cell-penetrating peptides, and several other methods.

One strategy to deliver unstable, anionic nucleic acids into cells and protect them from protease degradation is by encapsulating the cargo in lipids. Lipid nanoparticles (LNPs) are synthetic nanoparticles primarily made up of ionizable lipids, while extracellular vesicles (EVs) are membrane-derived lipid envelopes released naturally from cells that contain bioactive molecules. Both can be given a payload of CRISPR cargo to deliver into cells. LNPs in particular are popular for in vivo CRISPR delivery; they were widely adopted during the COVID-19 pandemic to deliver mRNA payloads in vaccines.

• Advantages: LNPs and EVs have minimal safety and immunogenicity concerns due to lack of viral components, and in the case of EVs, they are derived from human cells. All three types of CRISPR cargo can be delivered using these methods. The last several years have seen significant progress in the development of organ-targeted LNPs. For example, selective organ targeting (SORT) nanoparticles are engineered LNPs attached to a SORT molecule, making them targeted to specific cell types within lung, spleen and liver tissues.
• Disadvantages: A key issue associated with LNPs is the internal cellular processes that occur after delivery. They are usually encased by endosomes, and must ‘escape’ to avoid being directed into the lysosomal pathway and degraded, as well as gain access to the nucleus of the cell. In contrast, EVs have strong potential for tissue-homing but their inherent complexity and heterogeneity have so far hindered their clinical translation.

Another method for allowing nucleic acids to gain entry into cells is by complexing them with cationic lipids (lipoplexes) or cationic polymers (polyplexes). Although less popular than LNPs, these delivery vehicles are still being actively investigated. For a more detailed breakdown, you can read this review of gene delivery by lipoplexes and polyplexes.

• Advantages: Similar to other non-viral methods, there is less chance of associated severe immune responses when utilizing lipoplexes and polyplexes.
• Disadvantages: As described for liposomes, lipoplexes and polyplexes must escape from endosomes and gain entry to the nucleus of the cell. Various reagents have different transfection efficiencies, with some being better suited for use with certain types of cells.

Other types of nanoparticles (NPs) for CRISPR-Cas9 delivery are also being investigated for their potential in CRISPR delivery. They are made up of inorganic materials, including gold (AuNPs), silica, and magnetic particles.

• Advantages: NPs can deliver all three types of CRISPR cargo into cells. While each type of NP will have varying qualities, they generally have high stability and efficiency, relatively low cost, reduced off-target effects, high loading capacity, and lack of immune response and mutagenesis. They are an appealing vehicle for in vitro and in vivo studies.
• Disadvantages: Some NPs tend to aggregate in a physical medium. AuNPs have a key disadvantage of being toxic at high concentrations, and since they are relatively new methods, there are some concerns regarding the long-term toxicity and accumulation of other NPs, such as magnetic NPs.

Another non-viral CRISPR delivery method is cell-penetrating peptides (CPPs). CPPs are short, positively charged peptides that facilitate the uptake of molecules by the cell. They are usually conjugated with the CRISPR cargo via covalent bonds.

• Advantages: CPPs can be used for both in vitro and ex vivo CRISPR studies. They can also be used for many different types of cells, including primary human T cells, and can achieve high genome editing efficiencies. There is potential for CPPs to be modified to target specific cell or tissue types.
• Disadvantages: CPPs do not provide the cargo protection that encapsulation techniques like LNPs do. Although recent research has demonstrated their use in animal models, it is unclear if they can be successfully translated to in vivo genome editing in humans.

Physical CRISPR delivery methods include techniques such as electroporation, microinjection, and microfluidics. These delivery methods are typically only suitable for editing in ex vivo cell therapies and in vitro studies. Electroporation is the most common physical CRISPR delivery method and recent technological advances have made it an attractive avenue for the development of gene-edited cell therapies.

This technique involves opening transient pores in the membrane of cells via high-voltage electrical currents, allowing CRISPR cargo to gain entry. Electroporation also includes nucleofection, which is a more specialized method that delivers the cargo to the nuclei of cells.

• Advantages: Electroporation can be used for many different cell types, including those that are otherwise difficult to transfect. It can also deliver all three types of CRISPR cargo of any size, and is appropriate for both in vitro and ex vivo studies. This method of CRISPR delivery also avoids the use of viral vectors, which can be preferable for clinical applications.
• Disadvantages: Historically, electroporation has not always been suitable for every experiment or cell type. Mammalian cells, for example, are sensitive to electrical currents and many electroporation methods can change the cell state and compromise cell viability. However, Maxcyte’s scalable, cGMP compliant electroporation technology has enabled the successful CRISPR editing of primary human cells, including T cells, without reducing cell viability. This makes it an attractive CRISPR delivery method for the development of cell therapies.

This less-common CRISPR delivery method involves using microscopic channels to manipulate fluids, making the cell membrane transiently permeable so that cargo can be delivered into cells.

• Advantages: This technique is gentle on cells, and has been demonstrated to be effective when editing primary human immune cells, such as T cells and natural killer cells.
• Disadvantages: Microfluidics has not yet been widely adopted for CRISPR delivery. Many microfluidic chips can only accommodate relatively small numbers of cells, as the arrays of the chip can become clogged with cells.

To learn more, you can check out our interview with Ryan Pawell, CEO of Indee Labs, who discusses µVS and his goal of providing scalable and affordable CRISPR-edited T-cells for immunotherapy.

Microinjection delivers CRISPR cargo into individual cells by piercing the membrane with a microscopic needle.

• Advantages: This CRISPR delivery method can deliver cargo of any molecular weight, at known quantities, to either the cytoplasm or nucleus of the cell. It is most often used for delivery of nucleic acid cargo, and has high efficiency. When used to deliver RNA cargo, it will have restricted duration within the cells, typically reducing off-target effects.
• Disadvantages: This method can be a challenging and laborious process. It is limited to animal models and the modification of cells ex vivo, and is not suitable for in vivo studies.