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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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society of Gene & Cell Therapy
2023
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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. |
format | Online Article Text |
id | pubmed-10025986 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Society of Gene & Cell Therapy |
record_format | MEDLINE/PubMed |
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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