Innovations in Gene Editing: Use of Cas9 Viral Particles in CRISPR Delivery

To fully exploit the gene editing potential of CRISPR/Cas9, they must be efficiently introduced into target cells or tissues using appropriate vectors. Recombinant viral vectors have been developed using ability of viruses to transfer foreign genetic material into cells to deliver therapeutic genes to diseased tissues. Among many viral vectors, adeno-associated virus (AAV), lentivirus, and adenovirus play a crucial role in genome editing therapy and have been widely used in preclinical models and clinical trials. Although modified viral vectors do not cause severe human disease, they can induce immune system-mediated clearance, which may reduce delivery efficiency. Another feature of viral vectors is the ability to integrate DNA into the host genome to achieve stable gene expression, which may lead to off-target effects and insert mutation. Therefore, the application of virus delivery methods is sophisticated. This article will review the merits and defects of each delivery method.

Adenovirus

Adenovirus is a double-stranded DNA virus with a diameter of 80-100 nm. Its genome is ~ 34-43 kb in length and can package ~ 8 kb of exogenous DNA. Due to its excellent ability to carry large genetic cargo, delivery efficiency of the adenovirus vector-mediated CRISPR/Cas9 module can be improved by conferring additional nuclear localization signals. Continuous advancement of technology has generated adenoviral vectors lacking the viral genome, allowing loading of target DNA up to 37 kb.

GEMMs of human cancer are important tools to analyze the molecular mechanisms of tumorigenesis. Introducing CRISPR/Cas9 into somatic cells of adult animals using adenovirus vectors induces specific chromosomal rearrangements to generate a mouse model of Eml4-Alk-driven lung cancer. This strategy expands how scientists simulate human cancer in model organisms by simplifying complex and time-consuming genetic manipulations.

Fig. 1 Induction of Eml4–Alk rearrangement in murine cells using the CRISPR-Cas9 system. (Maddalo, et al., 2014)

Lentivirus

Lentivirus is a subcategory of the retrovirus family, and the lentivirus genome contains a single-stranded RNA of 7-12 kb. Lentiviral vectors provide effective cell transduction in various cell types (including dividing and non-dividing cells) and shorten the culture time required for cell transfection. Compared with adenovirus or AAV vectors, lentivirus shows low cytotoxicity and immunogenicity and has minimal impact on transduced cells. Because of their relative ease of use, lentiviruses are promising as in vivo delivery systems. Normally, lentivirus integrates its genome into the host genome, which can significantly extend the time for transgene expression. However, continuous expression of Cas9 may increase the risk of off-target effects and hinder application in high-precision genome editing. As an alternative, integration-deficient lentiviral vectors generated by integrase mutation can greatly reduce the risk of insertion mutations.

Preclinical studies show that lentiviral delivery Cas9 and guide RNA targeting mutated KRAS significantly inhibits proliferation of cancer cells. Further, lentiviral delivery of CRISPR/Cas9 targeting BCR-ABL significantly inhibits myelogenous leukemia cell growth and tumorigenesis, so therapies based on ABL gene editing may provide a potential strategy for imatinib-resistant chronic myeloid leukemia patients.

Adeno-associated virus (AAVs)

AAV vectors have many advantages, such as lack of pathogenicity, long-term gene expression, and the ability to infect dividing and non-dividing cells, so they are used extensively for in vivo delivery systems. In addition, AAV family is characterized by rich serotype diversity and has variable tropism, specifically targeting different organs. Although AAVs are excellent gene therapy delivery vehicles, they still have weaknesses when used to deliver CRISPR/Cas9 in vivo. The optimal AAV vector size is 4.1-4.9 kb. Although AAV can package vectors larger than its genome size, packaging efficiency drops sharply. Another major problem of AAV is pre-existing neutralizing antibodies against AAV in patients with previous AAV infection, which greatly reduces therapeutic efficacy. However, combining capsid modification and genome modification to produce an optimized AAV serotype vector can reduce affinity with neutralizing antibodies, thereby reducing host immune response and improving delivery efficiency. In addition, long-term transgene expression of AAV also may be a risk, because continuous expression of Cas9 nuclease may cause significant off-target effects.

Although there are still many challenges to overcome, people have begun to explore AAV-mediated CRISPR delivery. The AAV dual-vector system successfully targets a single gene or multiple genes in the mouse brain and characterizes the influences of genome modification on neurons, suggesting that that AAV-mediated genome editing can be applied to study brain gene function.

Development of safe and effective in vivo delivery remains the biggest challenge for widespread clinical use of CRISPR/Cas9 in human therapy. Most current clinical studies use viral vectors, but challenges such as immunogenicity, cytotoxicity and carcinogenicity still need to be conquered.

CRISPR/Cas9 PlatformCB, with years of experience in gene editing research, provides our customers with efficient Cas9 virus particles, providing an effective method to drive high levels of Cas9 nuclease expression in various cell types.

CATALOG NO.	PRODUCT NAME
CCV-001	Cas9 Puromycin Lentivirus Particles
CCV-002	Cas9 Blasticidin Lentivirus Particles
CCV-003	Cas9 Puromycin Adenovirus Particles
CCV-004	Cas9 Blasticidin Adenovirus Particles

References

Wang, S. W.; et al. Current applications and future perspective of CRISPR/Cas9 gene editing in cancer. Molecular cancer. 2022, 21(1): 1-27.
Maddalo, D.; et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature. 2014, 516(7531): 423-427.
Kim, W.; et al. Targeting mutant KRAS with CRISPR-Cas9 controls tumor growth. Genome research. 2018, 28(3): 374-382.

* For research use only. Not intended for any clinical use.

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Innovations in Gene Editing: Use of Cas9 Viral Particles in CRISPR Delivery

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