title: 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.
  
  
  
  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.
authors.affiliation: Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota, Minneapolis, Minnesota cjbierle@umn.edu.
authors.forename: Craig J
authors.identifier: http://orcid.org/0000-0002-7890-7409
authors.initials: CJ
authors.lastname: Bierle
authors.affiliation: Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota, Minneapolis, Minnesota.
authors.forename: Kaitlyn M
authors.initials: KM
authors.lastname: Anderholm
authors.affiliation: Department of Pediatrics, Virginia Commonwealth University School of Medicine, Richmond, Virginia.
authors.forename: Jian Ben
authors.initials: JB
authors.lastname: Wang
authors.affiliation: Department of Pediatrics, Virginia Commonwealth University School of Medicine, Richmond, Virginia.
authors.forename: Michael A
authors.initials: MA
authors.lastname: McVoy
authors.affiliation: Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota, Minneapolis, Minnesota.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Schleiss
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial; D003587:Cytomegalovirus / Q000235:genetics* / Q000378:metabolism*; D003586:Cytomegalovirus Infections / Q000235:genetics / Q000821:virology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D006168:Guinea Pigs; D008168:Lung / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D017393:RNA Editing / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
chemical_list: D012367:RNA, Viral