Multiplex CRISPR/Cas9 system impairs HCMV replication by excising an essential viral gene

PLoS One. 2018 Feb 15;13(2):e0192602. doi: 10.1371/journal.pone.0192602. eCollection 2018.


Anti-HCMV treatments used in immunosuppressed patients reduce viral replication, but resistant viral strains can emerge. Moreover, these drugs do not target latently infected cells. We designed two anti-viral CRISPR/Cas9 strategies to target the UL122/123 gene, a key regulator of lytic replication and reactivation from latency. The singleplex strategy contains one gRNA to target the start codon. The multiplex strategy contains three gRNAs to excise the complete UL122/123 gene. Primary fibroblasts and U-251 MG cells were transduced with lentiviral vectors encoding Cas9 and one or three gRNAs. Both strategies induced mutations in the target gene and a concomitant reduction of immediate early (IE) protein expression in primary fibroblasts. Further detailed analysis in U-251 MG cells showed that the singleplex strategy induced 50% of indels in the viral genome, leading to a reduction in IE protein expression. The multiplex strategy excised the IE gene in 90% of all viral genomes and thus led to the inhibition of IE protein expression. Consequently, viral genome replication and late protein expression were reduced by 90%. Finally, the production of new viral particles was nearly abrogated. In conclusion, the multiplex anti-UL122/123 CRISPR/Cas9 system can target the viral genome efficiently enough to significantly prevent viral replication.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Cytomegalovirus / genetics
  • Cytomegalovirus / physiology*
  • Flow Cytometry
  • Humans
  • Polymerase Chain Reaction
  • Virus Replication*

Grant support

This work was supported by the Labex IGO project (n° ANR-11-LABX-0016-01) funded by the «Investissements d’Avenir» French Government program, managed by the French National Research Agency (ANR) (; a grant from the Fondation Centaure(; a research grant from the European Society of clinical Microbiology and Infectious Diseases (ESCMID)(, a Junior basic grant from the European Society of Organ transplantation (ESOT) (