# 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
#### 3.2.3 CRISPR Systems Targeting Epstein-Barr Virus  
The Application of CRISPR Systems to Edit EBV Latency-Associated Genes  
Simultaneously targeting three latency-associated genes involved in maintaining viral episomes and/or host cell transformation (i.e., _EBV nuclear antigen 1_ or _EBNA1_, _EBNA3C_, and _latent membrane protein 1_ or _LMPI_) and three structural repeat regions (Pstl repeats, EBNA-leader protein (LP) repeats, and 125 bp repeats) in EBV-infected Raji cells with a mixture of seven gRNAs and Cas9, administered via plasmid transfection, resulted in a decrease in EBV load, reaching almost complete elimination in a quarter of the cell population. It also resulted in complete suppression of cell proliferation and a decrease in total cell count, indicating the initiation of cell apoptosis [46]. These findings suggest that CRISPR-Cas9 editing of the EBV genome can restore the apoptotic pathway previously interrupted by EBV [46]. While targeting only the repeat regions led to a complete suppression of cell proliferation, only a small decrease in EBV load was observed. Vice versa, targeting the EBNA1 coding region strongly reduced the viral load without completely suppressing cell proliferation [46]. This was confirmed by another study [63] using a different infection model. Targeting the _EBNA1_ coding region, _latent origin of replication_ (_OriP_), or W repeats individually with EBV-specific CRISPR-Cas9-encoding plasmids reduced virion production in HEK293 cells carrying the M81 EBV strain. In latently EBV-infected nasopharyngeal carcinoma (NPC) cells, viral DNA loads decreased progressively over a period of 4 weeks post-transfection with CRISPR-Cas9 plasmids targeting _EBNA1_, _OriP_, and/or W repeats. However, in all experiments, the CRISPR system failed to completely eradicate the EBV episomes or alter cell viability and proliferation, even when the dose was increased. Still, the NPC cells were sensitized to chemotherapeutic killing following CRISPR treatment [63]. Targeting _LMPI_ or _LMP2A_ by transfection of EBV-CRISPR-Cas9 plasmids in another type of EBV-infected NPC cells (Cantonese nasopharyngeal epithelial cells or CNE-2 cells) inhibited EBV replication [64]. Even cell growth was significantly reduced upon _LMPI_ targeting [64]. Disruption of _LMPI_ in a third type of EBV-infected NPC cell line (NPC C666-1) by another CRISPR-Cas9 delivery method (i.e., poly[\(\beta\)-amino ester] or PBAE-plasmid polyplexnanoparticles [NPs]) also reduced tumor cell viability, cell growth, and the ability to form colonies [65]. In vivo, the delivery of these CRISPR-Cas9 complex NPs in murine EBV-NPC xenograft tumor models significantly inhibited tumor growth and induced tumor cell apoptosis, with tumor inhibition rates up to 45-81% [65; 39]. Finally, lentiviral transduction of [PERSON]'s lymphoma Akata-Bx1 cells with a singleplex CRISPR-Cas9 system targeting _EBNA1_ or _OriP_ resulted in the loss of viral gene transcription and EBV genomes in approximately half the cells [45]. Sequential transduction with two lentiviral vectors carrying different gRNAs against _EBNA1_ and/or _OriP_ strongly enhanced these inhibitory effects on virus replication. The highest efficiency was noted upon double targeting of _EBNA1_, as more than 95% of the cells were cleared from the EBV genome [45].  
The Application of CRISPR Systems to Induce the Reactivation of Latent EBV Infection  
One study [66] applied CRISPRRa to target the EBV IE gene _Z trans-activator (ZTA)_ promoter region in EBV-positive B and epithelial cancer cells via lentiviral transduction to achieve EBV reactivation and consequently sensitize the cells to ganciclovir treatment. Almost all CRISPR-dCas9-gRNA-expressing EBV-positive cells in each cell line succumbed following ganciclovir treatment. Similar results were observed in EBV-positive gastric cancer Seoul National University (SNU)-719 cells through plasmid transfection for more transient CRISPR delivery [66].