# CRISPR-Cas Systems: Prospects for Use in Medicine
## 6 Potential Challenges in Using CRISPR-Cas Systems  
Despite the relatively high efficiency of CRISPR-Cas as a tool for genome editing, it has a number of potential disadvantages and risks. These include, in particular, the use of the NHEJ, leading to a high probability of mutations (see above), and the off-target effect also resulting in editing mistakes [57; 134; 135]. Though the frequency of such off-target mutations is quite low, allowing to use CRISPR-Cas systems for plants and microorganisms genome editing, they nevertheless represent the most serious problem in gene therapy, since they can lead to unpredictable short-term and long-term consequences [136]. These limitations can be overcome by ex vivo gene therapy approaches [137], while in vivo genome editing in humans, especially fetal gene therapy and manipulations with germ line cells, may still raise ethical questions [138].  
The Cas9 nuclease, as mentioned earlier, possesses its own cytotoxic effect on certain microorganisms [139]. These difficulties can be overcome by using endogenous nucleases, when working with those microorganisms, which possess them. Endogenous nucleases are also used when working with genomes of extremophilic bacteria or archaea, since mesophilic bacterial nucleases do not work well in these cells [140].  
Another problem of using CRISPR-Cas, especially when using them as antibacterial agents, is their delivery. RNA-guided Cas nucleases targeting plasmid-located virulence and drug-resistance genes can be delivered to microbial populations using bacteriophages or bacteria carrying conjugative plasmids.  
Phage delivery systems are a promising tool in the fight against drug resistance, but they have several disadvantages. Despite the difficulties encountered by bacteria in developing resistance to the injected CRISPR-Cas system itself, they can easily develop phage resistance. A narrow species-specificity of a phage, that on the one hand makes it a convenient tool for CRISPR-Cas delivery to a specific microorganism, on the other hand limits its use for other bacteria, thus a search for various phage-delivery systems is required.  
As phages themselves are the natural targets of CRISPR-Cas systems, some of them have anti-CRISPR proteins (Acrs) that target various components of the effector complexes. They are usually highly-specific to certain CRISPR-Cas systems [141; 142; 143]. The anti-CRISPR proteins are relatively small (not exceeding 150 amino acid residues). The expression of genes encoding these proteins is regulated by the Aca1 transcription factor, the presence of which is mandatory for all anti-CRISPR systems. The mechanism of action of this system has not yet been fully elucidated, and only six anti-CRISPR proteins have been studied. AcrF1 and AcrF2 form a complex with Cas-proteins and RNA, preventing its binding to phage DNA. AcrF3 inhibits Cas3 activity, also by forming a non-functional complex. AcrIIC1 inhibits the Cas9\({}_{\text{Spy}}\) nuclease, which can be implied in gene surgery and engineering to regulate CRISPR-Cas activity in certain tissues or at certain stages of the organism's life cycle [143].