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CRISPR/CasRx Proof-of-Concept for RNA Degradation: A Future Tool against RNA Viruses?
Influenza viruses provide a great threat for the human population, causing highly contagious respiratory infections that can lead to serious clinical complications. There are a limited variety of influenza antivirals, and these antivirals are subjected to the constant emergence of resistances. There...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8778981/ https://www.ncbi.nlm.nih.gov/pubmed/35056089 http://dx.doi.org/10.3390/ph15010032 |
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author | Perez-SanJose, Diana de la Fuente, Miguel Angel Serna Pérez, Julia Simarro, Maria Eiros Bouza, José María Sanz-Muñoz, Ivan |
author_facet | Perez-SanJose, Diana de la Fuente, Miguel Angel Serna Pérez, Julia Simarro, Maria Eiros Bouza, José María Sanz-Muñoz, Ivan |
author_sort | Perez-SanJose, Diana |
collection | PubMed |
description | Influenza viruses provide a great threat for the human population, causing highly contagious respiratory infections that can lead to serious clinical complications. There are a limited variety of influenza antivirals, and these antivirals are subjected to the constant emergence of resistances. Therefore, the development of new antiviral strategies to combat influenza viruses and other RNA viruses must be promoted. In this work, we design a proof-of-concept of a recently described CRISPR/Cas tool that has been proposed as a possible future RNA virus antiviral, named CRISPR/CasRx. For this, we verified the efficiency of the CasRx endonuclease in the degradation of the eGFP mRNA reporter gene and we established the best conditions for, and the efficient performance of, the CRISPR/CasRx system. The results were measured by fluorescence microscopy, flow cytometry, and qRT-PCR. The analyses demonstrated a reduction in fluorescence, regardless of the amount of eGFP reporter plasmid transfected. The analyses showed an 86–90% reduction in fluorescence by flow cytometry and a 51–80% reduction in mRNA expression by qRT-PCR. Our results demonstrate that the CasRx endonuclease is an efficient tool for eGFP mRNA knockdown. Therefore, subsequent experiments could be useful for the development of a new antiviral tool. |
format | Online Article Text |
id | pubmed-8778981 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-87789812022-01-22 CRISPR/CasRx Proof-of-Concept for RNA Degradation: A Future Tool against RNA Viruses? Perez-SanJose, Diana de la Fuente, Miguel Angel Serna Pérez, Julia Simarro, Maria Eiros Bouza, José María Sanz-Muñoz, Ivan Pharmaceuticals (Basel) Article Influenza viruses provide a great threat for the human population, causing highly contagious respiratory infections that can lead to serious clinical complications. There are a limited variety of influenza antivirals, and these antivirals are subjected to the constant emergence of resistances. Therefore, the development of new antiviral strategies to combat influenza viruses and other RNA viruses must be promoted. In this work, we design a proof-of-concept of a recently described CRISPR/Cas tool that has been proposed as a possible future RNA virus antiviral, named CRISPR/CasRx. For this, we verified the efficiency of the CasRx endonuclease in the degradation of the eGFP mRNA reporter gene and we established the best conditions for, and the efficient performance of, the CRISPR/CasRx system. The results were measured by fluorescence microscopy, flow cytometry, and qRT-PCR. The analyses demonstrated a reduction in fluorescence, regardless of the amount of eGFP reporter plasmid transfected. The analyses showed an 86–90% reduction in fluorescence by flow cytometry and a 51–80% reduction in mRNA expression by qRT-PCR. Our results demonstrate that the CasRx endonuclease is an efficient tool for eGFP mRNA knockdown. Therefore, subsequent experiments could be useful for the development of a new antiviral tool. MDPI 2021-12-27 /pmc/articles/PMC8778981/ /pubmed/35056089 http://dx.doi.org/10.3390/ph15010032 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Perez-SanJose, Diana de la Fuente, Miguel Angel Serna Pérez, Julia Simarro, Maria Eiros Bouza, José María Sanz-Muñoz, Ivan CRISPR/CasRx Proof-of-Concept for RNA Degradation: A Future Tool against RNA Viruses? |
title | CRISPR/CasRx Proof-of-Concept for RNA Degradation: A Future Tool against RNA Viruses? |
title_full | CRISPR/CasRx Proof-of-Concept for RNA Degradation: A Future Tool against RNA Viruses? |
title_fullStr | CRISPR/CasRx Proof-of-Concept for RNA Degradation: A Future Tool against RNA Viruses? |
title_full_unstemmed | CRISPR/CasRx Proof-of-Concept for RNA Degradation: A Future Tool against RNA Viruses? |
title_short | CRISPR/CasRx Proof-of-Concept for RNA Degradation: A Future Tool against RNA Viruses? |
title_sort | crispr/casrx proof-of-concept for rna degradation: a future tool against rna viruses? |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8778981/ https://www.ncbi.nlm.nih.gov/pubmed/35056089 http://dx.doi.org/10.3390/ph15010032 |
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