Targeted Mutagenesis of Guinea Pig Cytomegalovirus Using CRISPR/Cas9-Mediated Gene Editing

Abstract

The cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome in Escherichia coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates. IMPORTANCE The cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infection in vivo.Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virus-infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation.

CRISPR/Cas9‐mediated 2‐sgRNA cleavage facilitates Pseudorabies virus editing

“…Several reports have used transfection-infection-based methods to knock out PRV genes, with knockout ratios ranging from 12.5 to 42.9% (14,19). Another large DNA viral genome was reportedly used as part of a strategy to efficiently introduce targeted deletions by this method (37). In that study, CRISPR-mediated gene editing of guinea pig cytomegalovirus was evaluated (37).…”

“…Another large DNA viral genome was reportedly used as part of a strategy to efficiently introduce targeted deletions by this method (37). In that study, CRISPR-mediated gene editing of guinea pig cytomegalovirus was evaluated (37). The authors first transfected CRISPR constructs for 48 h and then added puromycin to kill the untransfected cells.…”

CRISPR/Cas9, a powerful tool to target human herpesviruses

“…Studies on the use of CRISPR/Cas9 to target cytomegaloviruses are limited to two recent reports (Bierle, Anderholm, Wang, McVoy, & Schleiss, ; van Diemen et al, ). Bierle et al () showed that by transient transfection of CRISPR/Cas9 constructs carrying anti‐GP133 gRNAs, indel mutations could be introduced at the guinea pig cytomegalovirus target site, thereby inactivating the gene.…”

“…Studies on the use of CRISPR/Cas9 to target cytomegaloviruses are limited to two recent reports (Bierle, Anderholm, Wang, McVoy, & Schleiss, ; van Diemen et al, ). Bierle et al () showed that by transient transfection of CRISPR/Cas9 constructs carrying anti‐GP133 gRNAs, indel mutations could be introduced at the guinea pig cytomegalovirus target site, thereby inactivating the gene. Additionally, the authors could direct the deletion of large segments of the viral gene by co‐transfection of two gRNAs, and they succeeded in epitope tagging of GP133 by HDR upon co‐delivery of a gRNA and donor template carrying an Human influenza hemagglutinin (HA)‐tagged version of the gene (Bierle et al, ).…”

“…Bierle et al () showed that by transient transfection of CRISPR/Cas9 constructs carrying anti‐GP133 gRNAs, indel mutations could be introduced at the guinea pig cytomegalovirus target site, thereby inactivating the gene. Additionally, the authors could direct the deletion of large segments of the viral gene by co‐transfection of two gRNAs, and they succeeded in epitope tagging of GP133 by HDR upon co‐delivery of a gRNA and donor template carrying an Human influenza hemagglutinin (HA)‐tagged version of the gene (Bierle et al, ). These studies clearly show the potential of the CRISPR/Cas9 technology to allow efficient genetic manipulation of CMV isolates.…”