Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR/Cas9
- Harvard Medical School, United States
- Harvard University, United States
- Harvard T H Chan School of Public Health, United States
Abstract
Herpes simplex virus (HSV) establishes lifelong latent infection and can cause serious human disease, but current antiviral therapies target lytic but not latent infection. We screened for sgRNAs that cleave HSV-1 DNA sequences efficiently in vitro and used these sgRNAs to observe the first editing of quiescent HSV-1 DNA. The sgRNAs targeted lytic replicating viral DNA genomes more efficiently than quiescent genomes, consistent with the open structure of lytic chromatin. Editing of latent genomes caused short indels while editing of replicating genomes produced indels, linear molecules and large genomic sequence loss around the gRNA target site. The HSV ICP0 protein and viral DNA replication increased the loss of DNA sequences around the gRNA target site. We conclude that HSV, by promoting open chromatin needed for viral gene expression and by inhibiting the DNA damage response, makes the genome vulnerable to a novel form of editing by CRISPR-Cas9 during lytic replication.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files.
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Human herpesvirus 1 isolate KOS, complete genomeNCBI GenBank: KT899744.1.
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hg38Genome Reference Consortium, Human GRCh38.p12 (GCA_000001405.27).
Article and author information
Author details
Werner M Neuhausser
Funding
National Institutes of Health (P01 AI098681)
- David M Knipe
National Institutes of Health (R21 AI135423)
- Kevin C Eggan
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Oh et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR/Cas9
eLife 8:e51662.
https://doi.org/10.7554/eLife.51662
Further reading
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- Genetics and Genomics
One of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs. Such circuits bridge the properties of electronic and natural circuits, processing information in a predictable manner within living cells. Genome editing is a potentially powerful component of synthetic molecular circuits, whether for modulating the expression of a target gene or for stably recording information to genomic DNA. However, programming molecular events such as protein-protein interactions or induced proximity as triggers for genome editing remains challenging. Here, we demonstrate a strategy termed ‘P3 editing’, which links protein-protein proximity to the formation of a functional CRISPR-Cas9 dual-component guide RNA. By engineering the crRNA:tracrRNA interaction, we demonstrate that various known protein-protein interactions, as well as the chemically induced dimerization of protein domains, can be used to activate prime editing or base editing in human cells. Additionally, we explore how P3 editing can incorporate outputs from ADAR-based RNA sensors, potentially allowing specific RNAs to induce specific genome edits within a larger circuit. Our strategy enhances the controllability of CRISPR-based genome editing, facilitating its use in synthetic molecular circuits deployed in living cells.
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