July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
CRISPR/Cas9-Mediated Genome Editing of Herpesviruses Limits Productive and Latent Infections
Author Affiliations & Notes
  • Saskia Imhof
    Ophthalmology, UMC Utrecht, Utrecht, Netherlands
  • Footnotes
    Commercial Relationships   Saskia Imhof, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 512. doi:https://doi.org/
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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. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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