Potential CRISPR-Cas9-based Antiviral Activity Against Hepatitis C Virus in Liver Cancer

Hepatitis C virus (HCV) is a primary global health concern, and though therapeutic options have improved, no very effective vaccine is available despite decades of research. The health and vitality of the organism are related to the result of homeostatic regulation of the internal environment. In contrast, it follows that disruption of homeostatic mechanisms leads to disease, particularly in liver cancer. Since HCV can rapidly mutate to evade the immune response, an effective HCV vaccine must rely on the identification and characterization of sites critical for broad immune protection and viral neutralization. In this work, we discussed the Hepatitis C virus (HCV) from the viewpoint of homeostasis due to its heterogeneous structure with numerous genomes and different recognized subunits containing discrete segments. However, the interplay between HCV proteins during genomic RNA replication and progeny virion assembly is not completely understood. Here, we studied the dynamics and intracellular localization of non-structural 5A protein (NS5A), a protein involved in genome replication. HCV genome can encode several proteins with about 4,000 amino acids, each containing glycoproteins (E1 and E2) and seven non-structural (NS) proteins. E1 and E2 form a heterodimer complex on the virus structure, where E2 contains a receptor binding domain (RBD) that affects entry receptors. Since DNA double-strand breaks (DSB) often involve breaks in either foreign DNA strand, it can be repaired using NHEJ or HDR pathways. By this method, through enzyme endonuclease, site-specific segments of DNA are inserted, and then DNA repair mechanisms are used to close the DNA breaks, known as genetic scissors (CRISPR/Cas9). CRISPR/Cas9 technology is precise and rapidly reprogrammable depending on the experimental setup. Therefore, specific genomic loci can be activated or interfered with by CRISPR/Cas9 ribonucleic-protein (CRISPR or CREPR) interactions. This NS5A-APEX2 protein was fully visualized by fast transmission electron microscopy (TEM). These results exhibited hepatitis virus (HCV) replication during conjugated membrane vesicles containing replication complexes. Most studies on NS5A dynamics highlight the increased movement of NS5A in the absence of the nucleus, which could be related to altered nucleus-dependent activity associated with microtubules and/or dynein.