# CRISPR-Cas Systems: Prospects for Use in Medicine
## 3 Genome Editing
### Mechanism of Genome Editing  
Genomic editing systems based on Cas9 nuclease are currently the most widely used, since this system requires only this protein, in contrast to complex multiprotein complexes that are necessary for the functioning of other CRISPR systems. Cas9 from _Streptococcus pyogenes_ (Cas9\({}_{\text{Spy}}\)) is the most frequently used Cas9 nuclease. However, in some cases, _Streptococcus thermophilus_ nuclease is also used. Cas9 nuclease contains two functional domains--HNH and RuvC. The first one cleaves the target DNA strand, while the second domain cuts the complementary strand [40]. The CRISPR-Cas9 is a class 2, type II system. It includes the Cas9 nuclease, the trans-activating CRISPR RNA (tracrRNA), and crRNA. crRNA is formed during the processing of precrRNA, transcribed from the CRISPR cassette. precrRNA is cut into crRNAs, consisting of spacers and their adjacent DRs. crRNA in complex with tracrRNA guides Cas9 (the so-called interference module) to target specific sequences on the exogenous DNA. crRNA and tracrRNA can be fused into a chimeric single guide RNA (sgRNA) [41; 42; 43]. The specificity of targeting in this case is determined by both the complementarity of sgRNA and the target DNA, and by PAM motifs. The complex also requires RNase III processing for its function. The ability to use sgRNAs instead of separate crRNAs and tracrRNAs is more convenient for genome editing and represents another advantage of Cas9.  
The Cas9/sgRNA complex scans the DNA sequence until PAM is found. Then DNA uncoiling occurs in the so-called priming region (10 bp immediately after the PAM) [44]. If the DNA sequence is complementary to the sgRNA sequence, the Cas9 HNH-domain introduces a break in the target strand, and the RuvC-domain--in the non-target strand [45, 46]. Thus, CRISPR-Cas9 is capable of introducing double-stranded breaks (DSBs) at target sites in genomic DNA, resulting in a gap [47].  
The introduced gap must be repaired afterwards. In eukaryotic genome editing techniques, this may be done by the non-homologous end-joining (NHE). In this case, the cut DNA ligation occurs without a homologous DNA matrix, either directly or with the introduction of an insertion or deletion. This method has a number of drawbacks, primarily consisting in the significant frequency of insertions and deletions leading to frameshifts and a loss of gene functionality [48]. However, this property of NHEJ can be used to target gene knockout. NHEJ can be also used to completely remove a target gene.  
Another mechanism is homologous directed repair (HDR), which involves replacing a deleted sequence with a new sequence complementary to a specifically introduced DNA template. Its advantage is high accuracy, while its disadvantage is low frequency (less than 20%, while NHEJ is up to 60%) [49]. The search for ways of increasing HDR frequency is very important for further development of this area. HDR can be used both for introducing and reverting target mutation in the genome, and for inserting whole genes in the genome using a template [50, 51].  
One of the drawbacks of HDR consists of occasional NHEJ repair that occurs despite the presence of the template. Specialized NHEJ inhibitors are used to override this, but they can have a cytotoxic effect [52, 53].