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...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-12-01
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| Series: | Pharmaceuticals |
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| Online Access: | https://www.mdpi.com/1424-8247/15/1/32 |
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| author | Diana Perez-SanJose Miguel Angel de la Fuente Julia Serna Pérez Maria Simarro José María Eiros Bouza Ivan Sanz-Muñoz |
| author_facet | Diana Perez-SanJose Miguel Angel de la Fuente Julia Serna Pérez Maria Simarro José María Eiros Bouza Ivan Sanz-Muñoz |
| author_sort | Diana Perez-SanJose |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-10T00:45:39Z |
| format | Article |
| id | doaj.art-f5ff961e29a343cf9434dbcef3ddc815 |
| institution | Directory Open Access Journal |
| issn | 1424-8247 |
| language | English |
| last_indexed | 2024-03-10T00:45:39Z |
| publishDate | 2021-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Pharmaceuticals |
| spelling | doaj.art-f5ff961e29a343cf9434dbcef3ddc8152023-11-23T15:01:02ZengMDPI AGPharmaceuticals1424-82472021-12-011513210.3390/ph15010032CRISPR/CasRx Proof-of-Concept for RNA Degradation: A Future Tool against RNA Viruses?Diana Perez-SanJose0Miguel Angel de la Fuente1Julia Serna Pérez2Maria Simarro3José María Eiros Bouza4Ivan Sanz-Muñoz5National Influenza Center of Valladolid, Hospital Clínico Universitario de Valladolid, University of Valladolid, 47010 Valladolid, SpainTargeted Gene Modification Laboratory, Unidad de Excelencia Instituto de Biología y Genética Molecular (IBGM), 47003 Valladolid, SpainTargeted Gene Modification Laboratory, Unidad de Excelencia Instituto de Biología y Genética Molecular (IBGM), 47003 Valladolid, SpainTargeted Gene Modification Laboratory, Unidad de Excelencia Instituto de Biología y Genética Molecular (IBGM), 47003 Valladolid, SpainNational Influenza Center of Valladolid, Hospital Clínico Universitario de Valladolid, University of Valladolid, 47010 Valladolid, SpainNational Influenza Center of Valladolid, Hospital Clínico Universitario de Valladolid, University of Valladolid, 47010 Valladolid, SpainInfluenza 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.https://www.mdpi.com/1424-8247/15/1/32CRISPR/Cas systemantiviralinfluenza virusCRISPR/Cas13dCRISPR/CasRx |
| spellingShingle | Diana Perez-SanJose Miguel Angel de la Fuente Julia Serna Pérez Maria Simarro José María Eiros Bouza Ivan Sanz-Muñoz CRISPR/CasRx Proof-of-Concept for RNA Degradation: A Future Tool against RNA Viruses? Pharmaceuticals CRISPR/Cas system antiviral influenza virus CRISPR/Cas13d CRISPR/CasRx |
| 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 | CRISPR/Cas system antiviral influenza virus CRISPR/Cas13d CRISPR/CasRx |
| url | https://www.mdpi.com/1424-8247/15/1/32 |
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