Genome à la carte. How does CRISPR-Cas9 work?

CRISPR technology mimics the CRISPR-based immune system of some bacteria when viruses infect them. The mechanism discovered in bacteria works as follows: a bacterial protein capable of cutting DNA (Cas9) detects the viral genome with the help of a virus specific RNA (gRNA) guide and destroys it. This system also has a ‘memory’, like our immune system, since the gRNA used for a given virus can be stored in the genome of the bacteria for further infections.

And how does this allow us to edit the genome?

The two main characters of this story are the guide RNA (composed by a scaffold part and a genome specific part); and the DNA ‘scissors’, Cas9. To start with, both the guide RNA and the Cas9 are ‘fed’ to the cell. The RNA guide is designed in the lab to target the DNA region to be modified. Once inside, these two parts find each other and the RNA guide acts as a GPS helping Cas9 to find out the region of interest in the genome (figure 1 and 2). After arriving, Cas9 cuts the DNA. The cut forces the DNA repair machinery of the cell, a sort of cellular firefighter, to move into this area. During the repair processes, and depending on the final objective, two different scenarios may occur (figures 3 and 4):

• Gene silencing or Knock-Out (KO) (Figure 4.1). It is the simplest way to use CRISPR-Cas9 and the objective is to remove existent information in the genome. The repair machinery pastes together the remaining DNA in the cutting site, although causing mutations in the original sequence. This results in an inactivation of the gene.
• DNA insertion or Knock-In (KI) (Figure 4.2). This happens when the goal is to add extra information to the genome. In this case, a third actor enters the scene: the ‘repair template’, which will be introduced in the cell together with Cas9 and the guide. This repair template is a piece of DNA that contains the information that will be added (in purple), surrounded by homology regions (in orange) that bind to the region of interest. In this context, the repair machinery will recognize this repair template and will add it to the genome in the position that the homology regions indicate.

What is the impact of this breakthrough on our society?

The three main areas of application of this technique are: research, agriculture & plant biotechnology, and medicine. The most common use is in research, where scientists modify genes of interest for their studies, such as genes involved in determined diseases. Another implementation is the production of transgenic plants. What is the advantage here? The production of plants that are resistant to certain viruses, for example. However, the implementation of this technology for plant modification is not yet legal in some countries, as is the case for the European Union countries.

And last but not the least, the use of CRISPR-Cas9 for medical purposes. Gene therapy by using this tool is probably the most exciting and hoped-for application of this technology. Through it, a new type of treatments for several diseases would be possible (genetic and infectious diseases, cancers, etc.). Although some of these therapies are already in clinical trials, off-target effects, challenges on how to deliver the components, immunogenicity, and ethical issues do still pose problems for their implementation. Once solved, the incorporation of CRISPR-Cas9 into our lifes will be a matter of time!