# Applying CRISPR Technologies for the Treatment of Human Herpesavirus Infections: A Scoping Review
## 3 Results
### Overview of CRISPR Strategies Used to Target Different Human Herpesviruses
More precisely, lentiviral transduction or plasmid transfection of Vero cells with a CRISPR-Cas9 system simultaneously targeting _UL8-UL29_, _UL8-UL52_, _UL29-UL52_, or _UL19-UL30_ inhibited viral replication, even reaching an HSV-1-free Vero cell population in one study [41; 45; 54]. Additionally, double gene-targeting of _UL8-UL29_ or _UL29-UL52_ in HSV-1-infected Medical Research Council strain 5 (MRC5) cells via lentiviral delivery of a CRISPR-Cas9 system resulted in the complete loss of infectious particles [45]. In line with the IE and E genes, targeting the HSV-1 L genes impaired virus replication. For instance, targeting the L HSV-1 _gD_ gene, encoding the gD envelope glycoprotein, using a CRISPR-gRNA-Cas9 plasmid significantly reduced HSV-1 titers in human embryonic kidney (HEK293)-AD cells [55]. Additionally, lentiviral transduction of Vero cells with a CRISPR-Cas9 system targeting _UL15_, _UL27_, _UL36_, or _UL37_ resulted in impaired HSV-1 replication [45]. Interestingly, even CRISPR-Cas9-mediated knock-out of nonessential genes (_unique short 3_ [_US3_] or _US8_) or non-coding regions (intergenic regions between _UL26-UL27_ and UL37-UL38)_ was able to reduce HSV-1 replication in Vero cells and human foreskin fibroblasts, respectively [45; 56]. This reduction is likely due to defects in virus particle assembly arising from unrepaired dsDNA breaks, as the rapid HSV-1 replication kinetics exceed cellular DNA repair capacities, resulting in impaired infection of naive cells [45]. Instead of viral genes, one study [61] targeted the host gene _double homeobox 4 (DUX4)_, of which the translated protein is involved in the transcription activation of HSV-1 E and L genes upon stimulation by HSV-1 IE proteins. Indeed, lentiviral transduction of a haploid human (HAP1) cell line and HEK 293T cells with a CRISPR-Cas9 system against _DUX4_, followed by HSV-1 infection, impaired HSV-1 replication. As it was observed that all human herpesviruses induce the expression of DUX4, DUX4 knock-out could potentially impair the replication of other herpesviruses as well [61]. Lastly, [PERSON] et al. [60] took an approach of multi-point genome editing by designing a strategy named coordinated lifecycle elimination against viral replication (CLEAR). CLEAR is based on the simultaneous editing of IE (_ICP4_ and _ICP27_) and L (_VP16_, _gD_) genes, both active during different steps in the virus life cycle. Targeting each gene individually upon plasmid delivery of the CRISPR system to baby hamster kidney (BHK)-21 cells, followed by HSV-1 infection, significantly reduced the viral titer, noting the highest and lowest efficiency upon targeting _ICP4_ and _VP16_, respectively. This antiviral effect was doubled when all genes were targeted simultaneously in the CLEAR strategy. In case BHK-21 cells were first infected with HSV-1 prior to CLEAR treatment, virus inhibition was the most prominent in the early phase post-transfection, suggesting a small time window of less than 12 h for treatment. In vivo, intracerebral injection of lentiviruses carrying the CRISPR-Cas9 system in murine prevention models reduced viral replication at the injection site and prevented viral transmission to downstream brain areas. These antiviral effects were more prominent with the CLEAR strategy compared to individual _ICP4- or ICP27_-targeting. In line with the prevention model, HSV-1 proliferation and transmission were significantly inhibited in murine treatment models, receiving the treatment 1 day post-HSV-1 infection [60]. Simultaneous editing of one or more E and L genes was also achieved using a unique delivery mechanism. More precisely, [PERSON] et al. [59] designed autonomous, intelligent, virus-inducible immune-like (ALICE) systems that can autonomously detect intracellular pathogens and subsequently activate antiviral responses.