The CRISPR/Cas gene editing system is widely applied in animals, plants, and microorganisms. Since delivery efficiency varies depending on the characteristics of the target organism, including differences between species as well as between cell and tissue types within the same species, selecting an appropriate delivery strategy is essential. Here we take human cells as an example and outline commonly used delivery methods developed in recent years, along with their strengths, limitations, and application scenarios.

This approach transfects the plasmids carrying gRNA sequences and Cas protein coding regions into target cells via liposomes or electroporation.

Strengths:

a. Straightforward and rapid experimental workflow.

b. The high capacity vector allows both gRNA and Cas sequences to be cloned into one single plasmid.

Limitations:

a. Transfection efficiency varies by cell type. Primary cells and stem cells often show low efficiency.

b. Transfection reagents or electroporation may reduce cell viability.

Application Scenarios: Non-viral plasmids are easy to store and amplify. This strategy is widely used in basic research and high-throughput screening in easy-to-transfect cell lines.

In this approach, the gRNA plasmid and Cas9 construct are separately packaged into lentiviral particles and then transduced into target cells for long-term expression.

Strengths:

a. Lentivirus can efficiently infect most cell types.

b. The gene editing cassettes are integrated into the host genome, enabling stable, long-term expression.

Limitations:

a. Limited packaging capacity requires gRNA and Cas proteins to be packaged and delivered separately.

b. Editing onset is delayed due to the lentiviral lifecycle (reverse transcription, integration, and expression).

c. The cassettes are integrated into host genome randomly, which may disrupt essential genes.

d. Infection efficiency remains low in certain primary cells and immune cells.

Application Scenarios: Lentivirus is often employed as a supplementary approach for gene editing in difficult-to-transfect cells. Since CRISPR/Cas9 can induce heritable mutations at genomic level, the benefit of long-term stable expression from lentiviral vectors is relatively limited in the gene editing field. Nonetheless, lentivirus remains a valuable tool for establishing stable Cas9 expressing cell lines. Once Cas9 is stably expressed, only gRNA needs to be transfected, making gene editing faster, simpler, and more consistent.

Combining the strengths of lentiviral systems and CRISPR/Cas technology, EDITGENE has successfully generated stable Cas9 expressing cell lines for a wide range of cell types, providing researchers with a robust and efficient platform for gene editing.

AAV particles are used to package and deliver gRNA and Cas expression constructs into target cells.

Strengths:

a. Its low immunogenicity makes AAV widely used in gene therapy.

b. Tissue tropism varies by serotypes. When combined with tissue-specific promoters, AAV approach allows precise in vivo editing in target tissues.

c. AAV persists as an episome in the cell, allowing long-term expression with minimal risk of genomic integration.

Limitations:

a. Limited packaging capacity of AAV requires smaller Cas9 options, such as SaCas9 or split-Cas9. However, SaCas9 restricts gRNA design due to its PAM constraints, whereas split-Cas9 typically exhibits lower efficiency.

b. In dividing cells, episomal genomes become diluted during cell proliferation, shortening the expression duration.

Application Scenarios: AAV is commonly used in animal studies and in vivo gene therapy, particularly suitable for terminally differentiated, non-dividing tissues (such as brain, eye, liver, muscle), taking full advantage of its low immunogenicity and high tissue specificity.