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Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene

DNA-targeting CRISPR-Cas systems are able to cleave dsDNA in mammalian cells. Accordingly, they have been employed to target the genomes of dsDNA viruses, mostly when present in cells in a non-replicative state with low copy numbers. However, the sheer amount of viral DNA produced within a very shor...

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Autores principales: Didara, Zrinka, Reithofer, Florian, Zöttl, Karina, Jürets, Alexander, Kiss, Izabella, Witte, Angela, Klein, Reinhard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Gene & Cell Therapy 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10025986/
https://www.ncbi.nlm.nih.gov/pubmed/36950281
http://dx.doi.org/10.1016/j.omtn.2023.02.033
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author Didara, Zrinka
Reithofer, Florian
Zöttl, Karina
Jürets, Alexander
Kiss, Izabella
Witte, Angela
Klein, Reinhard
author_facet Didara, Zrinka
Reithofer, Florian
Zöttl, Karina
Jürets, Alexander
Kiss, Izabella
Witte, Angela
Klein, Reinhard
author_sort Didara, Zrinka
collection PubMed
description DNA-targeting CRISPR-Cas systems are able to cleave dsDNA in mammalian cells. Accordingly, they have been employed to target the genomes of dsDNA viruses, mostly when present in cells in a non-replicative state with low copy numbers. However, the sheer amount of viral DNA produced within a very short time by certain lytically replicating viruses potentially brings the capacities of CRISPR-Cas systems to their limits. The accessibility of viral DNA replication sites, short time of accessibility of the DNA before encapsidation, or its complexation with shielding proteins are further potential hurdles. Adenoviruses are fast-replicating dsDNA viruses for which no approved antiviral therapy currently exists. We evaluated the potency of CRISPR-Cas9 in inhibiting the replication of human adenovirus 5 in vitro by targeting its master regulator E1A with a set of guide RNAs and observed a decrease in infectious virus particles by up to three orders of magnitude. Target DNA cleavage also negatively impacted the amount of viral DNA accumulated during the infection cycle. This outcome was mainly caused by specific deletions, inversions, and duplications occurring between target sites, which abolished most E1A functions in most cases. Additionally, we compared two strategies for multiplex gRNA expression and obtained comparable results.
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spelling pubmed-100259862023-03-21 Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene Didara, Zrinka Reithofer, Florian Zöttl, Karina Jürets, Alexander Kiss, Izabella Witte, Angela Klein, Reinhard Mol Ther Nucleic Acids Original Article DNA-targeting CRISPR-Cas systems are able to cleave dsDNA in mammalian cells. Accordingly, they have been employed to target the genomes of dsDNA viruses, mostly when present in cells in a non-replicative state with low copy numbers. However, the sheer amount of viral DNA produced within a very short time by certain lytically replicating viruses potentially brings the capacities of CRISPR-Cas systems to their limits. The accessibility of viral DNA replication sites, short time of accessibility of the DNA before encapsidation, or its complexation with shielding proteins are further potential hurdles. Adenoviruses are fast-replicating dsDNA viruses for which no approved antiviral therapy currently exists. We evaluated the potency of CRISPR-Cas9 in inhibiting the replication of human adenovirus 5 in vitro by targeting its master regulator E1A with a set of guide RNAs and observed a decrease in infectious virus particles by up to three orders of magnitude. Target DNA cleavage also negatively impacted the amount of viral DNA accumulated during the infection cycle. This outcome was mainly caused by specific deletions, inversions, and duplications occurring between target sites, which abolished most E1A functions in most cases. Additionally, we compared two strategies for multiplex gRNA expression and obtained comparable results. American Society of Gene & Cell Therapy 2023-03-03 /pmc/articles/PMC10025986/ /pubmed/36950281 http://dx.doi.org/10.1016/j.omtn.2023.02.033 Text en © 2023 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Original Article
Didara, Zrinka
Reithofer, Florian
Zöttl, Karina
Jürets, Alexander
Kiss, Izabella
Witte, Angela
Klein, Reinhard
Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene
title Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene
title_full Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene
title_fullStr Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene
title_full_unstemmed Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene
title_short Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene
title_sort inhibition of adenovirus replication by crispr-cas9-mediated targeting of the viral e1a gene
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10025986/
https://www.ncbi.nlm.nih.gov/pubmed/36950281
http://dx.doi.org/10.1016/j.omtn.2023.02.033
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