RNA-guided gene editing of the murine gammaherpesvirus 68 genome reduces infectious virus production.
Animals
B-Lymphocytes
/ virology
CRISPR-Cas Systems
/ genetics
Cell Line
Gammaherpesvirinae
/ genetics
Gene Editing
/ methods
Gene Expression Regulation, Viral
/ genetics
Genome, Viral
/ genetics
HEK293 Cells
Herpesviridae Infections
/ virology
Herpesvirus 8, Human
/ genetics
Humans
Mice
Models, Animal
NIH 3T3 Cells
RNA, Guide, Kinetoplastida
/ genetics
Virus Activation
/ genetics
Virus Latency
/ genetics
Virus Replication
/ genetics
Journal
PloS one
ISSN: 1932-6203
Titre abrégé: PLoS One
Pays: United States
ID NLM: 101285081
Informations de publication
Date de publication:
2021
2021
Historique:
received:
01
03
2021
accepted:
13
05
2021
entrez:
4
6
2021
pubmed:
5
6
2021
medline:
25
2
2023
Statut:
epublish
Résumé
Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.
Identifiants
pubmed: 34086743
doi: 10.1371/journal.pone.0252313
pii: PONE-D-21-06827
pmc: PMC8177658
doi:
Substances chimiques
RNA, Guide
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, N.I.H., Intramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e0252313Subventions
Organisme : NIAID NIH HHS
ID : R43 AI131958
Pays : United States
Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
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