CRISPR/Cas9 Delivery Methods

This subpage constitutes the fourth part of the theory for Biotech Academy’s material on CRISPR-Cas9.

In order to perform genetic modification, Cas9 and sgRNA must be introduced into cells through the cell membrane and enter the cell nucleus into the genomic DNA. This delivery can be done by different approaches. Cas9 can be introduced as coding DNA, mRNA or finished protein. sgRNA can be introduced directly into cells in RNA form, or can be encoded as DNA. If an insertion or replacement of a gene sequence is to be performed, the DNA template must also be introduced. The different methods by which the genetic modification system can be delivered have different advantages and disadvantages, and one must therefore choose the delivery method that best suits the given situation. The delivery of the genetic modification system can be done by designing a vector containing all components of the system. Such a vector is based on genetic material which has the function of coding the components and being able to deliver them together to the cells in which the modification is to take place.

DNA-based delivery: Plasmids

It is possible to combine Cas9-coding DNA (cas9 gene) and sgRNA-coding DNA in a plasmid, which is a circular piece of DNA. This brings the entire system together in a single piece of DNA. It can also encode a DNA template. These plasmids act as vectors for the genetic modification system and can be produced by recombinant DNA technology. If the plasmid is transferred to an organism, it is expressed and thus the genetic modification system will be formed and become active. The components are expressed by inserting promoters in front of the coding sequences in the plasmid, which are regulatory sequences that result in the transcription of the DNA. These plasmids are designed specifically after the organism that you want to genetically modify, as different organisms have different ways of expressing DNA. The promoters must thus also function in the given organism in order for them to express the genetic modification system from the plasmid. The plasmid should not be replicant, otherwise it may copy itself into many specimens, which would be unnecessary for the gene modification and may be unfavorable to the cell.
DNA-based delivery by plasmid is illustrated in Figure 10. The plasmid must generally be delivered to the cell nucleus, where the components are constantly transcribed into RNA, i.e. Cas9-coding mRNA and sgRNA. Here, the plasmid can be stable for a longer period. The mRNA is translated into Cas9 protein in the cytoplasm, after which it forms complex with the selected sgRNA. Eventually, Cas9 penetrates the cell nucleus, where the genetic modification of the genomic DNA will take place.

RNA-based delivery

This is a delivery of Cas9 coding mRNA and sgRNA. Here, the mRNA simply needs to be translated into the finished Cas9 protein in the cytoplasm, after which it can form complex with sgRNA. It may be advantageous that mRNA is more unstable than DNA, as the translation of Cas9 is thus only short-lived. The dose of Cas9 can thus be regulated more than with DNA-based delivery, as a plasmid will continue to form new mRNA, which will produce more Cas9.
RNA-based delivery is illustrated in Figure 10. The RNA is delivered to the cytoplasm, where the Cas9-coding mRNA is translated. Subsequently, the Cas9 protein can form complex with sgRNA and penetrate the cell nucleus and perform the genetic modification, as in DNA-based delivery.

 

Protein-based delivery

A direct delivery of the finished Cas9 in complex with sgRNA means that the genetic modification can take place immediately after delivery, as there is no need for transcription or translation. This method has been shown to provide a more efficient and accurate gene modification, but it can be difficult to deliver a large protein compared to delivering RNA or DNA. Protein-based delivery provides the most short-term presence of Cas9, as the protein is not continuously formed from introduced RNA or DNA, which is thought to lower the risk of error and possibly decrease unwanted immunological responses.
Protein-based delivery is illustrated in Figure 10. The supplied Cas9 with sgRNA simply needs to penetrate the cell nucleus, in which the genetic modification is carried out, as with the other delivery methods.

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Figure 10. Different delivery methods for the genetic modification system. Both Cas9 and sgRNA must be delivered to the cell to be genetically modified, which can be done in several different formats; DNA, RNA or protein. Depending on the method, the cell’s own transcription and translation can be utilized to form the active Cas9 with gRNA.

Physical delivery methods

DNA-, mRNA- and protein-based delivery can be done by methods such as microinjection, electroporation, and packaging into nanoparticles. Microinjection is done by manual injection of the genetic modification system into a single cell, with a microscopic needle that can penetrate the cell membrane. Electroporation is a process in which cell membranes are made more permeable by exposing cells to an electric field. The genetic modification system can pass the membrane because it becomes unstable during electroporation. Nanoparticles that can pass through the cell membrane can be used to wrap the genetic modification system. For example, lipid nanoparticles containing the genetic modification system can be taken up by the cell by endocytosis.

Viral delivery method

There are viral vectors, which are harmless virus particles that can deliver genetic material to cells. Viruses specialize in infecting cells, which they do by binding to specific surface proteins of cells and introducing genetic material that takes over cell functions and ensures the reproduction of new virus particles.
Viral vectors are modified so that they are not harmful and cannot reproduce themselves. Instead, they are fed with self-selected genetic material that codes for sgRNA and Cas9. The viral vectors can still bind to the cells, and now transfer the self-selected genetic material, preferably without any side effects and immunological response. One challenge with viral vectors may be the lack of space in the virus particle for all the DNA you want to deliver, with which you have to plan how to package your virus. The most commonly used viral vector used for the delivery of Cas9 is adeno-associated virus (AAV).