How to choose the right CRISPR-Cas9 delivery method?

Just as important as good sgRNA design is the choice of a conducive to the introduction of the genome editing tool based on cell type that you want to modify in order to successfully achieve the change in the target gene. At In this sense, several formats for introducing nuclease are available, such as in the form of DNA, mRNA or protein with DNA transfection being the most (Yamamoto 2015). The following are the different approaches that are addressed for the transfection of nuclease and sgRNA either following biochemical, physical, viral or protein fusion methods. In the biochemical transfection of the carrier DNA of the components of the genomic editing, i.e. expression vectors, the researcher uses chemical reagents which have previously been recommended for use in  Genomic Engineering using CRISPR Systems-Cas López Mancheño, Y. A. 22 compared efficiencies by testing them in the study cells and with the vector of transfection control. This method is highly used and popular thanks to its good efficiency and its property of being scalable and adaptable to different conditions. Furthermore, is the most useful procedure if working with cells in culture or embryos.

If we are talking about physical transfection methods, there are also various options available such as electroporation, nucleofection and microinjection. This type of mechanism is chosen in the case of study cells which are difficult to transfect, such as stem cells or primary cells. If the chosen method is electroporation, a strategy by which electrical pulses determined by the type of cell are applied and create transitory pores in the cell membrane allowing DNA, RNA and proteins to be introduced, the most usual in relation to CRISPR-Cas9 is that Cas9 is introduced as a protein already purified together with sgRNA forming ribonucleoprotein structures of the machinery that makes up the tool already assembled. In order to facilitate the entry of ribonucleoproteins into the nucleus and thus increase the efficiency of the technique, nucleofection was developed after electroporation, in which, in addition to applying electrical pulses, it is bathed in a highly dampening solution of ionic force. Following the improvements to the initial technique (electroporation), from the 21st century onwards systems have been developed such as the neon transfection system which uses a pipette type electrode instead of a cell or new electroporators whose electrical pulses are poration (short and high voltage) and transfer (long and low voltage) to introduce the bioparticle of interest into the cells (Li and Hotta 2015).

The last physical method to be described, microinjection, consists in introducing normally DNA (in the case of CRISPR-Cas9 this DNA would be expression vectors) by means of a micro-needle of about 10 μm and a micromanipulator into the cell plasma or nucleus (Sakuma and Yamamoto 2015). Another form of transfection in relation to specific nucleases of design is transfection by virus. Viral vectors are tremendously efficient tools for introducing foreign genes into mammalian cells since this is the function they perform when they infect cells in their natural state. Several genomic editing works have been described by CRISPR-Cas9 using viral transfection vectors such as lentiviruses or adenoviruses (Abrahimi et al. 2015; Kabadi et al. 2014; Holkers et al. 2014). In addition to mammalian cells, in plants, Agrobacterium-mediated infection is usually used as an insertion method for CRISPR-Cas9 or, the transformation of protoplasts (Bortesi and Fischer 2014). Genomic Engineering using CRISPR-Cas López Mancheño, Y. A. Systems 23

Finally, the introduction of the tool can also be achieved through its conjugation with peptides with a penetrating capacity through the cell membrane In this case the purified Cas9 nuclease assembled with the relevant sgRNA forming ribonucleoproteins is chemically combined with the penetrating peptide which acts as a guide to cross the cell membrane. If this peptidibonucleoprotein fusion is simply added to the culture medium, it will penetrate the cells, although their entry can also be facilitated by nucleofection (Li and Hotta 2015).