Abstract:
UNLABELLED: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.
journal_name
J Viroljournal_title
Journal of virologyauthors
Bierle CJ,Anderholm KM,Wang JB,McVoy MA,Schleiss MRdoi
10.1128/JVI.00139-16subject
Has Abstractpub_date
2016-07-11 00:00:00pages
6989-6998issue
15eissn
0022-538Xissn
1098-5514pii
JVI.00139-16journal_volume
90pub_type
杂志文章abstract::We have studied the unintegrated infectious DNA of Harvey sarcoma virus (Ha-SV) and Moloney leukemia virus (Mo-MuLV). The source of infectious viral DNA was the Hirt supernatant fraction from cells acutely infected with Ha-SV and Mo-MuLV. To obtain a direct quantitative assay for infectious viral DNA, recipient mouse ...
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更新日期:1978-05-01 00:00:00
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pub_type: 杂志文章
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journal_title:Journal of virology
pub_type: 杂志文章
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pub_type: 杂志文章
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.55.3.840-842.1985
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pub_type: 杂志文章
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更新日期:1994-01-01 00:00:00
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更新日期:1979-08-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.65.10.5417-5424.1991
更新日期:1991-10-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.27.1.193-204.1978
更新日期:1978-07-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.63.9.3622-3633.1989
更新日期:1989-09-01 00:00:00
abstract:UNLABELLED:Lifelong antiretroviral therapy (ART) for HIV-1 does not diminish the established latent reservoir. A possible cure approach is to reactivate the quiescent genome from latency and utilize immune responses to eliminate cells harboring reactivated HIV-1. It is not known whether antibodies within HIV-1-infected...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.02717-15
更新日期:2015-12-09 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.40.3.625-634.1981
更新日期:1981-12-01 00:00:00
abstract::Three plaque isolates of SV40 strain 777 and 1 plaque isolate of strain 776 were grown to high-titer stocks and serially passaged, undiluted, in monkey BS-C-1 cells. In each case, the serial passaging procedure resulted in the accumulation of closed-circular SV40 DNA molecules containing covalently linked sequences ho...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.12.3.492-500.1973
更新日期:1973-09-01 00:00:00