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Saskia Imhof; CRISPR/Cas9-Mediated Genome Editing of Herpesviruses Limits Productive and Latent Infections. Invest. Ophthalmol. Vis. Sci. 2018;59(9):512.
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© ARVO (1962-2015); The Authors (2016-present)
HSV type 1 causes recurrent herpes simplex keratitis resulting in severe corneal scarring. Nucleoside analogues can efficiently limit the productive phase of a HSV infection. However, these drugs are ineffective preventing symptoms and scarring of the cornea. Genome engineering using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) holds potential to treat previously incurable HSV infections by directly targeting these viruses within infected cells. We investigated applications of the CRISPR/Cas9 system to treat productive HSV infections.
In the current study, we investigated whether direct targeting of the viral HSV-1 genome by CRISPR/Cas9 can be used to inactivate the virus within infected cells, thereby blocking virus replication. We generated a lentiviral CRISPR/Cas9 vector and asked whether human herpesviruses within infected cells can be genetically modified using this system. We designed 3-4 different gRNA’s targeting twelve essential HSV-1 genes and introduced these in human fetal lung MRC5 fibroblast cells. These cells were subsequently infected with HSV-1 carrying a green fluorescent protein (GFP) gene and virus replication was monitored by flow cytometry.
We show effective abrogation of HSV-1 replication by targeting CRISPR/Cas9 to essential viral genes. Simultaneous targeting of HSV-1 with multiple gRNAs completely abolished the production of infectious particles from human cells. The CRISPR/Cas9 system was active at limiting replicating virus, but appeared ineffective at targeting a latent HSV-1 infection in an in vitro HSV-1 quiescence model. However, virus replication upon HSV-1 reactivation from latency was efficiently abrogated.
We have shown that CRISPR/Cas9 technology can effectively impair HSV-1 replication in human cells. By combining two gRNAs targeting two essential HSV-1 genes, we completely inhibited the generation of new infectious virus particles during a productive HSV-1 infection in vitro. Our studies suggest that the CRISPR/Cas9 system may be used as a future prophylactic or therapeutic anti-viral strategy to interfere with HSV-1 infections in vivo.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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