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A knockdown of the herpes simplex virus type-1 gene in all-in-one CRISPR vectors
Affiliations- Cancer Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
- Department of Virology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
- Department of Pathobiology and Laboratory Sciences, School of Medicine, North Khorasan University of Medical Sciences, Bojnurd, Iran
- Department of Biology, Faculty of Sciences, University of Sistan and Baluchestan, Zahedan, Iran
- Department of Medical Biotechnology and Cancer Gene Therapy Research Center, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
- Department of Virology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
- Fuller Laboratories, Fullerton, CA, USA
- Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT, USA
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Abstract
Introduction. Herpes simplex virus type 1 (HSV-1) is a virus that causes serious human disease and establishes a long-term latent infection. The latent form of this virus has shown to be resistant to antiviral drugs. Clustered Regularly Interspace Short Palindromic Repeats (CRISPR), is an important tool in genome engineering and composed of guide RNA (gRNA) and Cas9 nuclease that makes an RNA-protein complex to digest exclusive target sequences implementation of gRNA. Moreover, CRISPR-Cas9 system effectively suppresses HSV-1 infection by knockout of some viral genes.
Materials and methods. To survey the efficacy of Cas9 system on HSV-1 genome destruction, we designed several guide RNAs (gRNAs) that all packaged in one vector. Additionally, we performed a one-step restriction using BamHI and Esp3I enzymes.
Results. CRISPR/Cas9 system targeted against the gD gene of HSV-1 was transfected into HEK-AD cells that showed a significant reduction of HSV-1 infection by plaque assay and real-time PCR.
Conclusion. The pCas-Guide-EF1a-GFP CRISPR vector can create a fast and efficient method for gRNA cloning by restriction enzymes (Esp3I (BsmBI) and BamHI). Therefore, the CRISPR/Cas9 system may be utilized for the screening of genes critical for the HSV-1 infection and developing new strategies for targeted therapy of viral infections caused by HSV-1.
Abstract
Introduction. Herpes simplex virus type 1 (HSV-1) is a virus that causes serious human disease and establishes a long-term latent infection. The latent form of this virus has shown to be resistant to antiviral drugs. Clustered Regularly Interspace Short Palindromic Repeats (CRISPR), is an important tool in genome engineering and composed of guide RNA (gRNA) and Cas9 nuclease that makes an RNA-protein complex to digest exclusive target sequences implementation of gRNA. Moreover, CRISPR-Cas9 system effectively suppresses HSV-1 infection by knockout of some viral genes.
Materials and methods. To survey the efficacy of Cas9 system on HSV-1 genome destruction, we designed several guide RNAs (gRNAs) that all packaged in one vector. Additionally, we performed a one-step restriction using BamHI and Esp3I enzymes.
Results. CRISPR/Cas9 system targeted against the gD gene of HSV-1 was transfected into HEK-AD cells that showed a significant reduction of HSV-1 infection by plaque assay and real-time PCR.
Conclusion. The pCas-Guide-EF1a-GFP CRISPR vector can create a fast and efficient method for gRNA cloning by restriction enzymes (Esp3I (BsmBI) and BamHI). Therefore, the CRISPR/Cas9 system may be utilized for the screening of genes critical for the HSV-1 infection and developing new strategies for targeted therapy of viral infections caused by HSV-1.
Keywords
CRISPR; Cas9; gRNA; genome editing; HSV-1; HEK-AD cells
About this articleTitle
A knockdown of the herpes simplex virus type-1 gene in all-in-one CRISPR vectors
Journal
Folia Histochemica et Cytobiologica
Issue
Article type
Original paper
Pages
174-181
Published online
2020-09-16
Page views
1796
Article views/downloads
1354
DOI
Pubmed
Bibliographic record
Folia Histochem Cytobiol 2020;58(3):174-181.
Keywords
CRISPR
Cas9
gRNA
genome editing
HSV-1
HEK-AD cells
Authors
Nastaran Khodadad
Mona Fani
Saleh Jamehdor
Rahil Nahidsamiei
Manoochehr Makvandi
Saeed Kaboli
Ali Teimoori
Jose Thekkiniath
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