How to choose the right CRISPR-Cas9 delivery method?

Currently, there are several methods for delivering CRISPR systems in vivo and in vitro. Both delivery vectors and physical methods are widely implemented for the efficient delivery of CRISPR/Cas9-mediated genome editing. Delivery vectors such as viral and non-viral vectors can accommodate mRNA or plasmid expressing the nucleases to target cells or tissues. Alternatively, physical methods including electroporation, laser, ballistic delivery, physical energy, or microinjection can be exploited for the delivery of nuclease into cells. Basically, non-viral vectors are preferable because of the limitations of viral vectors such as carcinogenesis, limited encapsulating capacity, and immunogenicity. Because of the low delivery efficiency in vivo, only a limited number of non-viral vectors for gene therapy have entered into clinical trials.

Over the years scientists have exploited viral vectors for their natural ability to penetrate into cells for successful gene delivery. For safety reasons, the pathogenic part of many viruses has been altered to function as carrier vehicles, including lentivirus, adenovirus and adeno-associated virus. One common feature between all viral mediated gene deliveries is the remarkable infection efficiency . Despite their notable infection efficiency, some viral vectors present the following drawbacks: (a) limited size of gene that can be delivered by the virus, (b) acute immune response associated with the viral vector and (c) production of viral vectors can be difficult

Physical Non-viral Gene Delivery

Physical methods, such as microinjection, electroporation and gene gun, uses physical force to disrupt the cell membrane in order to allow the gene to enter the cell. While these techniques are straightforward and easy to perform, specialized equipments are required for the procedures. In addition, since blunt force is applied, this method can cause tremendous cell death or tissue damage in some applications .

Chemical Non-viral Gene Delivery

Naked DNA molecules cannot enter the cell effectively due to their hydrophilic structure. Chemical methods utilize the electrostatic interactions between the negatively charged nucleic acid (i.e. DNA) with polycationic polymers or lipid particles to facilitate entry into the cells by endocytosis . The use of these chemical carriers offers three functions: (a) masks the negative charge on DNA, (b) compresses the DNA molecule into a smaller size and (c) protects the DNA from being degraded by intracellular nucleases. Apart from resulting in only short-term gene expression, the biggest disadvantage of chemical methods is that the delivery efficiency depends on the target cell type and cumbersome optimization is often required.