Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Perez-SanJose, Diana, de la Fuente, Miguel Angel, Serna Pérez, Julia, Simarro, Maria, Eiros Bouza, José María, Sanz-Muñoz, Ivan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
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
_version_ 1784637459819134976
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
work_keys_str_mv AT perezsanjosediana crisprcasrxproofofconceptforrnadegradationafuturetoolagainstrnaviruses
AT delafuentemiguelangel crisprcasrxproofofconceptforrnadegradationafuturetoolagainstrnaviruses
AT sernaperezjulia crisprcasrxproofofconceptforrnadegradationafuturetoolagainstrnaviruses
AT simarromaria crisprcasrxproofofconceptforrnadegradationafuturetoolagainstrnaviruses
AT eirosbouzajosemaria crisprcasrxproofofconceptforrnadegradationafuturetoolagainstrnaviruses
AT sanzmunozivan crisprcasrxproofofconceptforrnadegradationafuturetoolagainstrnaviruses