# Applying CRISPR Technologies for the Treatment of Human Herpesavirus Infections: A Scoping Review
## 4 Discussion
### Five Anti-Herpesviral Mechanisms of CRISPR Technologies  
A total of five mechanisms were identified in the application of CRISPR technologies to interfere with herpesvirus infection. The first mechanism involves targeting viral genes (IE, E, and/or L genes) or host genes to abrogate the viral lytic replication cycle and thus the active phase of the infection. Although this mechanism leads to the successful reduction of virus production, it is the least interesting option. Antiviral drugs targeting lytic herpesvirus infection already exist, and the small time window for treatment during the active phase of the infection would complicate realistic clinical implementation. A more interesting approach includes the application of CRISPR systems in latently infected cells, which comprises three possible mechanisms. The first mechanism, targeting genes important for latency maintenance, is the most promising since it could permanently cure infection by removing viral genomes from host cells, which has been impossible with the existing antiviral drugs. Given that latent EBV and KSHV proteins are involved in the oncogenesis of EBV- and KSHV-associated malignancies, evaluating the effects of CRISPR systems on latent EBV and KSHV gene expression is particularly interesting. It is important to note that, despite the efficient reduction in the number of latent viral genomes by the CRISPR-Cas9 and CRISPR systems, no study has achieved complete eradication of the latent EBV or KSHV episomes [38; 39; 44; 45; 46; 63; 64; 65; 72; 73]. This is likely due to multiple factors such as the presence of multiple viral genome copies per cell, incomplete delivery of CRISPR components, chromatin-associated protection of episomes, and the potential survival advantage of cells retaining episomes. The second mechanism, inhibiting viral reactivation by targeting lytic genes in latent viral genomes, could help prevent a flare-up of clinical symptoms and reduce the risk of virus transmission. Importantly, only a small editing efficiency of HSV-1 latent viral genomes and no reduction in the number of latent viral genomes was observed. Consequently, the question arises whether CRISPR systems can directly target lytic genes in the latent genomes and prevent reactivation or only target lytic genes when reactivation is induced and subsequently inhibit lytic replication. The latter theory could be supported by the hypothesis that gRNAs would be unable to reach the targeted sequences due to heterochromatinization of the viral genome during latency [9]. The third mechanism, purposefully inducing viral reactivation, can be used to sensitize the virus to the host immune system or antiviral drugs. This mechanism could be combined with the mechanism of targeting active lytic replication, thereby combining the antiviral effects of the host immune system, the current antiviral drugs, and CRISPR-Cas editing. Besides viral gene targets, host genes, such as _NETTIN-1_ and _DUX4_ for HSV-1, are an interesting choice as targeting the human genome instead of the viral genome could lessen the development of viral resistance. However, targeting sequences in the host genome poses biosafety risks as this could induce mutations in the human genome following NHEJ and must therefore be carefully evaluated prior to clinical implementation can be realized.  
A similar problem arises upon targeting the chromosomally integrated HHV-6A genomes as CRISPR-Cas9-mediated double-stranded cleavage in the host genome to excise the viral genome could induce pathological chromosomal rearrangements [80]. Similarly, host genes could be modified using CRISPR to generate EBV-specific cytotoxic T lymphocytes (CTLs) or modified virus-specific T cells (VSTs).