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

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

Author information

Centre de Recherche en Transplantation et Immunologie UMR 1064, INSERM, Université de Nantes, Nantes, France.
Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes, Nantes, France.
LabEx Immunograft Oncology (LABEX IGO), Nantes, France.
CIRI-International Center for Infectiology Research, Team EVIR, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Université Lyon, Lyon, France.
Institut für medizinische Mikrobiologie, und Krankenhaushygiene, Universität Marburg, Hans-Meerwein-Strasse 2, Marburg, Germany.
Inserm, U1065, Centre Méditerranéen de Médecine Moléculaire (C3M), équipe "contrôle métabolique des morts cellulaires", Nice, France.


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.

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