# 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
#### 3.2.6 CRISPR Systems Targeting [PERSON]'s Sarcoma-Associated Herpesvirus  
The Application of CRISPR Systems to Inhibit Lytic KSHV Replication  
Three studies [38; 71; 72] used the CRISPR-Cas system to edit lytic KSHV genomes. Targeting the IE gene _ORF57_ in high copy number KSHV-infected body cavity-based lymphoma (BCBL)-1 cells using two gRNAs and Cas9 delivered via a single expression vector reduced both viral genome copy numbers and lytic gene expression [71]. Knockout of _ORF57_ in human renal cell carcinoma inducible (i)SLK cells transfected with a KSHV bacterial artificial chromosome (BAC) clone (Bac16) did not result in viral genome instability, as the number of viral genome copies remained the same. However, upon lytic induction, the lytic viral replication was strongly attenuated as the _ORF57_ gene knockout had a detrimental effect on virion production [71]. CRISPR-Cas9-mediated targeting of the immediate early gene _ORF45_ induced INDELs and reduced KSHV DNA load in SLK cells [72]. Finally, CRISPR has been used to silence the expression of _ORF57_ or the delayed-early gene _ORF59_. Centrivial delivery of dCas9-Kruppel-associated box (KRAB) and gRNA targeting the promoter of _ORF57_ or _ORF59_ in KSHV latently infected iSLK-219 cells, followed by induced reactivation, led to ~90% knockdown of the expression of the respective gene. Consequently, targeting both genes substantially reduced KSHV lytic L gene (_K8.1_ gene) expression and viral titers [38]. The Application of CRISPR Systems Targeting KSHV Genes Important for  
Latency Maintenance  
Several studies [38; 72; 73; 44] applied CRISPR systems to target the expression of latency-associated nuclear antigen (LANA), a crucial KSHV latency protein encoded by _ORF73_. Transducing latently KSHV-infected Vero219 epithelial cells, L1T2 human endothelial cells, and human pleural effusion B lymphoblasts (BC3 cells) with an AAV5 encoding Cas9 and LANA-targeted gRNAs resulted in decreased LANA copy numbers and overall reduced KSHV episome burden [44]. The effects were time- and dose-dependent as they became more pronounced with time and increasing MOI. However, despite the loss of KSHV episomes, neither cell survival nor cell replication was affected [44]. In a different study [73], lentiviral delivery of the same CRISPR-Cas9 system in rat primary embryonic mesenchymal precursor cells (MM cells), which turn into tumor cells (KMM cells) following KSHV infection, resulted in no detectable expression of KSHV latency-associated genes, suggesting elimination of the viral episome. Importantly, KSHV episome elimination seemed to reverse the KSHV-mediated malignant transformation of the MM cells back to a normal state [73]. Transfection of BCBL-1 cells with liposome-encapsulated CRISPR-Cas9 ribonucleoprotein complexes targeting the LANA gene inhibited cell proliferation and induced cell apoptosis [73]. CRISPR-Cas9-mediated editing of the _ORF73_ gene decreased the KSHV DNA quantity in KSHV-infected SLK cells [72]. Lastly, lentiviral transduction of latently KSHV-infected iSLK-219 cells and BCBL-1 cells with a CRISPR system, consisting of dCas9-KRAB and gRNAs targeting the LANA promoter latent transcript (LTc) and/or LTi, substantially reduced LANA production in both cell lines and resulted in the loss of viral episomes in the transfected iSLK-219 cells [38]. The Application of CRISPR Systems to Induce the Reactivation of Latent KSHV Infection  
Two studies [40; 74] applied the CRISPR system to reactivate KSHV from its latent state into the lytic replication cycle. In a first study [40], activating the expression of the lytic IE _ORF50_ gene with a CRISPR system in KSHV-infected HEK293 cells resulted in increased viral genome copies, the expression of _ORF50_ and other IE, E, and L genes, and infectious virion production, indicative of full KSHV lytic replication cycle initiation. This effect was even more pronounced when two sites were targeted simultaneously [40].