## 3 Genome Editing via the CRISPR/Cas System  
As mentioned above, the latest and most promising tool for genome editing is the CRISPR/Cas system, short for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated gene (Cas) system [36; 37; 38]. The CRISPR/Cas system was originally developed by bacteria and archaea [39; 40; 41; 42; 43; 44] to identify an intruding virus and induce double strand breaks in its DNA [45]. The bacteria then incorporate this library of viral DNA fragments with which they have been infected into their genome as a defense mechanism. This library corresponds to the CRISPR sequences. In this way, when the bacteria are re-infected by the same virus, double-strand breaks are induced in the viral DNA by the Cas nuclease. The specific recognition and cutting of the viral genome by the Cas nuclease are reliant on two sequences: the guide RNA (gRNA), a 20-nucleotide (nt) sequence homologous to the target sequence, and the following protospacer adjacent motif (PAM), which is composed of 3-nt [36].  
Using the CRISPR/Cas system, we can theoretically target any DNA sequence. The only requirement is that the target sequence contains a PAM sequence. Each PAM sequence is recognized by the Cas nuclease of a particular bacterial species. The most commonly used is Cas9 from _Streptomyces pyogenes_ (SpCas9), which recognizes the PAM NGG. The PAM sequence in the target DNA sequence will dictate which Cas nuclease can be used. Once the Cas nuclease is delivered into the cells, it will induce a double-strand break in the target sequence. The cell will then repair the breaks [46; 47] using one of two main repair pathways: _non-homologous end-joining (NHEJ)_ or _homology-directed repair (HDR)_.  
During _NHEJ_, in order for the double-strand break to be repaired, the cell machinery joins the two ends of the break. However, this is an error-prone mechanism as insertions and deletions (indels) usually occur. These indels often induce a frameshift in the sequence, which would likely result in a premature termination codon. As a consequence, the corresponding gene is no longer functional [48]. This approach can be used to invalidate either the mutant allele specifically, if the gRNA only targets this allele, or both alleles (Figure 3). If Cas9 recognizes both alleles, then supplementation of the wild type gene is also required.  
During _HDR_, the cell uses the sister chromatid of the homologous chromosome as a template to repair the break. Alternatively, if a wild type sequence, usually in the form of a single-stranded oligodeoxynucleotide (ssODN), is provided to the cell simultaneously with the Cas9, that sequence can be used as a template to promote HDR repair [49]. This would be the method of choice in order to precisely correct an allele (Figure 3). The limitation of HDR is that it occurs much less frequently than NHEJ in the late S and G2 phases of cellular division [50]. Therefore, as the photoreceptors and the RPE are post-mitotic cells, they lack the HDR mechanism. A way to circumvent this issue has been proposed by [PERSON] and colleagues and is called HIT (Homology-Independent Targeted Integration).  
HITI uses the NHEJ mechanism, and not HDR, to integrate a DNA sequence. As a result, non-diving cells, such as photoreceptors, can be potentially edited using this method [51].  
Thus, taken together, the highly specific and efficient CRISPR/Cas system shows much potential for the treatment of the genetically heterogeneous IRD group [52].