*5.2. IE Gene Editing*

Instead of targeting IE gene expression at the mRNA level, genome-editing technology could be used to directly target the HCMV DNA genome to disrupt the UL122/123 gene responsible for major IE transcription. At the time of writing, one study has reported UL122/123 gene-editing, using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system [314]. CRISPR/Cas9 is a new powerful technology that targets specific DNA sequences in eukaryotic cells for cleavage, via double-stranded DNA breaks, using a Cas9 endonuclease and a guide RNA (gRNA) that determines target specificity. Double-stranded DNA breaks are repaired by host mechanisms, such as non-homologous end joining, which are error prone and can introduce small insertion/deletion mutations at the targeted location, or larger deletions if multiple breaks are introduced. A multiplex strategy using three gRNAs targeting UL122/123 successfully excised the UL122/123 gene in 90% of all viral genomes in an HCMV-infected cell population and resulted in a significant decrease in IE protein production and 90% reduction in HCMV replication [314]. Multiplex approaches have been developed to overcome acquisition of resistance mutations in the target sequences. This study provides proof-of-concept that a multiplex anti-UL122/123 CRISPR/Cas9 system can efficiently target the HCMV genome. This system may be useful for targeting HCMV in latently infected cells, where viral gene expression is low or absent and thus mRNA is not available for other molecular approaches discussed in this section and viral proteins (e.g., DNA polymerase) are not available as a target for conventional antiviral drugs.
