# 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  
Table 2, included at the end of the results section, provides an overview of CRISPR-based antiviral strategies against human herpesvirus and the respective targeted genes. As illustrated in Figure 2, it took only 2 years from the initial demonstration of CRISPR-Cas9 as a gene-editing tool in 2012 to its application as an antiherpesviral strategy. Over the following decade, different CRISPR systems have been developed and tested in vitro and in vivo to target six out of eight known human herpesvirus (Figure 2). These approaches are discussed in more detail below for each virus. Figure 1: PRISMA Flow Diagram, generated with the PRISMA Flow Diagram tool [36], schematically visualizing the article selection process. multiplex strategy, simultaneously targeting two sites of the same IE gene or two separate IE genes. Simultaneous transfection of Vero cells with two plasmids carrying Cas9 and gRNAs against different sites of _ICP0_ or _ICP27_, followed by HSV-1 infection, significantly reduced HSV-1 loads and titers [50]. This was further enhanced upon AAV2-mediated delivery of the CRISPR systems [50; 51]. Similar results were observed in a different study [42], as simultaneously targeting two genes, namely _ICP0_-_ICP4_, _ICP0_-_ICP27_, or _ICP4_-_ICP27_, in TC620 cells via lentiviral transduction of a CRISPR-Cas9 system resulted in the complete absence of HSV-1 infection. HSV-1 E genes are involved in viral DNA replication and have been targeted by multiple studies [41; 42; 45; 53; 54; 57; 58]. Delivery of a CRISPR-Cas9 system individually targeting the _unique long 5_ (_UL5_), _UL8_, _UL9_, _UL19_, _UL29_, _UL42_, or _UL52_ gene via lentiviral vectors or plasmid transfection into Vero cells and/or human foreskin fibroblasts significantly impaired HSV-1 replication in multiple studies [41; 45; 54; 56]. An alternative method for CRISPR-Cas delivery, namely engineered extracellular vesicles (EVs), was applied in a different study [58]. Delivery of engineered EVs carrying Cas9 and a gRNA against _UL29_, encoding ICP8, in HSV-1-infected Vero cells and/or Hela cells decreased _glycoprotein D_ (_gD_) and _viral protein 16_ (_VP16_) transcription, ICP8 and gD production, and the viral plaque number. Labeling the EVs with a neuro-targeting rabies virus glycoprotein peptide (RVG29) increased the targeting-specificity of the EVs toward nervous tissues both in vitro and in vivo. RVG-labeled EVs were even able to transverse endothelial cells to invade underlying neuronal hippocampal terminal (HT22) cells in vitro, indicating they could penetrate blood vessels. Administering the RVG-Cas9-UL29-gRNA EVs in vivo before HSV-1 infection significantly reduced the viral titer in the serum and resulted in less severe clinical symptoms and higher survival rates [58]. Four studies [41; 45; 54; 56] used a CRISPR-Cas9 system to target _UL30_, resulting in reduced HSV-1 replication in Vero cells and human foreskin fibroblasts. Besides INDELs in the HSV-1 genome, linear DNA species were also identified, indicating that CRISPR-Cas9 additionally inhibits HSV-1 by inducing large genome deletions and DNA degradation [56]. Besides CRISPR-Cas9, CRISPR-CasX targeting _UL30_ was also able to reduce HSV-1 infection in Vero cells [41]. A different E gene, namely _UL39_, encoding the ICP6 protein involved in viral replication, was targeted using the CRISPR-Cas9 system in two different studies [53; 57]. Delivery of Cas9 and anti-_UL39_ gRNAs substantially reduced viral loads in vitro in HSV-1-infected Vero cells following plasmid transfection and in vivo in brain tissues of HSV-1-infected Bagg albino laboratory-bred substrain c (BALB/c) mice upon topical eye application of the CRISPR therapy [53; 57]. Three studies [41; 45; 54] used a multiplex strategy to simultaneously target two E genes and observed higher antiviral effects compared to a singleplex strategy.