# CRISPR-Cas Systems: Prospects for Use in Medicine
## 4 CRISPR-Cas Implementation to Control Human Infectious Diseases
### Application of CRISPR-Cas to Combat Drug-Resistant Microorganisms  
CRISPRR-Cas has the potential to be used as antimicrobial agents for targeted cleavage of antibiotic resistance genes located on plasmids. When targeting drug resistance genes with plasmid localization, CRISPR-Cas systems cannot be considered a drug themselves. Nevertheless, such an approach, aimedto reduce the spread of drug-resistant strains, rather than to destroy the pathogen itself, makes sense, since an attempt to completely eliminate pathogenic microorganisms from their niches can lead to unpredictable consequences [30]. Drug-sensitive strains are put under the pressure of positive selection, and resistant ones, under negative selection. The main goal of introducing this technique is to remove drug resistance genes from the population and make it susceptible to drugs [114]. However, intended or accidental targeting of the bacterial genome sequence by the CRISPR-Cas system leads to cell death due to the appearance of irreversible chromosomal damage [115].  
In 2014 two groups showed that efficiently delivered RNA-guided nucleases can be used either for pathogen drug sensitization, or for its direct killing (depending on their targeting) both in vitro and in vivo [116; 117]. [PERSON] et al. used a CRISPR-Cas9 system delivered by the NM1 phage to destroy 99% of plasmids carrying antibiotic resistance genes, resulting in significant antibiotic sensitization of _Staphylococcus aureus_. Targeting genes on the chromosome had a bactericidal effect, which was also confirmed in vivo in the mouse skin colonization model [116]. The second study, reported by [PERSON] et al. implied two approaches to deliver the Cas9 nuclease targeting blaNDM-1 and blaSHV-18 (provide resistance to beta-lactam antibiotics): an M13-based plasmid and a conjugative plasmid. The use of a phage delivery system resulted in a 2-3-fold decrease in the number of viable enterohemorrhagic _E. coli_ cells. It was additionally shown that the CRISPR-Cas system could selectively target drug-resistant strains. A plasmid, targeting the _gyrA_ gene (responsible for fluoroquinolones resistance), was cytotoxic only for quinolone-resistant cells with a mutation in the _gyrA_ gene while being inactive against cells with the wild-type _gyrA_. This system worked as well in vivo in a _Galleria mellonella_ infection model [117].  
CRISPR-Cas may be considered an alternative to conventional antituberculosis chemotherapy. Currently administered drugs are effective against drug-susceptible _Mycobacterium tuberculosis_ strains; however, the number of drug-resistant tuberculosis cases is constantly growing. _M. tuberculosis_ lacks horizontal gene transfer and its drug resistance is mediated by mutations in genes encoding drug biotargets, activators of prodrugs, and efflux pumps' transcriptional regulators [118]. _M. tuberculosis_ possesses a subtype III-A CRISPR-Cas system (comprising the _cas1-cas2_ adaptation module, six _csm_ genes, _cas6_ gene responsible for crRNA processing, and two CRISPR cassettes), allowing an easy implementation of exogenous type II (Cas9) and type V (Cas12a) systems [119; 120; 121], furthermore a large number of known mycobacteria provides a good source for selecting specific and effective delivery systems [122].  
While RNA-guided antituberculosis agents have not yet been reported, several CRISPR systems were developed for mycobacteria, with the one based on _Streptococcus thermophilus_ dCas9 (dCas9\({}_{\text{Sth1}}\)) being the most efficient one [104]. These systems are mainly used for search of novel drug targets [123].  
Thus, CRISPR-Cas systems are emerging as powerful tools for combating the emergence of drug resistance. Their key features are high specificity, which allows them to be used to combat only a specific pathogen, without affecting the host microbiome, and the low chance of developing resistance to themselves due to the possibility of aiming several targets simultaneously.