## Genome editing technologies

As mentioned earlier, one of the greatest challenges in gene therapy is the precise editing of the genome. This was very difficult before the discovery of programmable nucleases. The term nuclease refers to a group of enzymes capable of cleaving nucleic acids. Of particular interest for gene therapy applications are nucleases that cause double-strand breaks in the DNA, such as the enzyme FokI, which is sequence-specific, meaning that it first recognizes a particular sequence in the genome and then cuts the DNA in that region. However, there are many such regions in the human genome, so the presence of this nuclease in the nucleus would mean fragmentation of the entire genome. This is obviously undesirable, so only the catalytic domain of this nuclease is retained and further linked to DNA-binding domains such as zinc finger (ZF) and transcription activator-like effector (TALE). What is important here is that we are able to modify the DNA-binding domains in such a way that they recognize a sequence of choice. The FokI nuclease functions as a dimer, meaning that two molecules of the enzyme must be present to form a functional complex. The DNA-binding domains must therefore be designed to bind on both sides of the site where the double-strand break is to be created (Fig. 10.8). This is very useful because the longer the recognition sequence, the lower the probability that the enzyme will accidentally cut the DNA in a different place in the genome as intended. An alternative to the aforementioned genome editing technologies is the CRISPR/Cas9 system. Similar to the examples already mentioned, it is a two-component system of a nuclease (Cas9) and a guiding molecule. These molecules are short RNAs (single guide RNA – sgRNA), which can be very easily modified so that, based on complementarity, they recognize practically any sequence in the genome. Together, Cas9 and sgRNA form a functional complex, which results in a double-strand break upon recognition of the target sequence (Fig. 10.8).

So the programmable nucleases allow us to create a double-strand break in the DNA molecule with high precision. In humans, there are two ways in which cells repair double-strand breaks (see Chapter 5). One of these methods is non-homologous end joining (NHEJ), in which the DNA ends are simply glued together (Fig. 10.8). However, errors in the form of small insertions or deletions occur relatively frequently. This can be useful when interrupting or removing the section of DNA that is the cause of the pathology. When a DNA template is supplied along with the nucleases, the breaks are repaired by a process known as homology directed repair (HDR). HDR enables DNA repair, insertion, and modification with very high precision and efficiency (Figure 10.8). Gene therapy with programmable nucleases therefore has great potential to make this type of treatment more precise and efficient but unfortunately we still have to wait for its entry into clinical practice.