# CRISPR-Cas Systems: Prospects for Use in Medicine
## 4 CRISPR-Cas Implementation to Control Human Infectious Diseases
### Application of CRISPR to Study Metabolic Pathways and Search for New Drugs' Biotargets  
It has been shown, that the introduction of two point mutations in Cas9 amino acid sequence (D10A and H840A) leads to a complete loss of endonuclease activity, while maintaining its ability to recognize and bind to target DNA sequence. This mutant is referred to as dCas9, or nuclease dead Cas9. Targeting dCas9 with specific sgRNAs to a gene's promoter region prevents the RNA polymerase from doing the same, thus interfering with the transcription. This approach was named CRISPR interference (CRISPRi) [103]. CRISPR can be used to conditionally knock-out almost any gene with high specificity. A simultaneous expression of different sgRNAs may be used for a knock-out of multiple genes [104]. Chimeric dCas9, fused to various transcription repressor domains, may be used to increase the efficiency of repression. The use of the Kruppel-associated box domain (KRAB domain) of the Kox1 protein was shown to provide the highest efficiency of transcription repression (up to 99%) [105; 106].  
CRISPRi can be used not only to downregulate, but also to upregulate a gene's expression. dCas9 can be fused to transcription activator domains to form the so-called CRISPR. CRISPR allows activating multiple genes by using multiple sgRNAs. Using multiple transcription activators simultaneously (synergistic activation mediators, SAM), such as VP64 (a tandem of four copies of the viral herpes simplex protein V16), p65 AD (transactivation domain of the NF-kB subunit p65), and Epstein-Barr virus transactivator R (Rta) (VPR) increases CRISPR efficiency [106; 107].  
Besides gene expression control, dCas9-based systems can also be used for genome editing. Hybrid dCas9, supplemented with modified adenosine deaminase, is able to point-convert A/T base pair to a G/C pair with a 50% efficiency. In this case, a non-functional nuclease is used for precise targeting, while adenosine deaminase is used directly for base modification without introducing DSBs [108].  
CRISPRi and CRISPR can also be used as genetic screening tools, especially in the development of new drugs, when it is necessary to establish the biotarget and possible factors mediating resistance for the candidate compounds selected during the initial screening [109]. CRISPR is a powerful tool for discovering new genetic pathways, functional genes' studies, including determining their role in drug resistance development. Cell screening using a random CRISPR library allows positive selection of cells in the population that have a fitness advantage, such as drug resistance. gRNAs from selected cells are analyzed to detect the genes conferring this advantage. In contrast, negative selection can result in finding vital genes-biotargets for further drug development [110].  
CRISPRi is an alternative to RNA interference (RNAi) based on double-stranded synthetic small RNA, complementary to a certain gene and designed to reduce the level of expression of this gene, widely used previously. The advantage of CRISPR is its ability to almost completely knock-down a gene's expression, while RNAi can only reduce it [111].  
CRISPRi systems are currently mainly used in prokaryotes to study metabolic pathways for novel biotargets' identification. However, it can also be used directly for disease treatment. In 2020 a doxycycline-induced system was successfully used to treat epilepsy in a rodent model. An increase in the expression of the Kcna1 potassium channel gene resulted in a significant decrease in neuronal excitability and, as a consequence, in relief of epilepsy symptoms [112]. Progress has been made in the immunotherapy of malignant diseases through the activation of several genes involved in an antitumor response (multiplexed activation of endogenous genes as an immunotherapy, MAEGI) [113].