open access

Vol 58, No 3 (2020)
Original paper
Submitted: 2020-07-29
Accepted: 2020-09-08
Published online: 2020-09-16
Get Citation

A knockdown of the herpes simplex virus type-1 gene in all-in-one CRISPR vectors

Nastaran Khodadad12, Mona Fani13, Saleh Jamehdor4, Rahil Nahidsamiei12, Manoochehr Makvandi12, Saeed Kaboli5, Ali Teimoori126, Jose Thekkiniath78
DOI: 10.5603/FHC.a2020.0020
·
Pubmed: 32937678
·
Folia Histochem Cytobiol 2020;58(3):174-181.
Affiliations
  1. Cancer Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
  2. Department of Virology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
  3. Department of Pathobiology and Laboratory Sciences, School of Medicine, North Khorasan University of Medical Sciences, Bojnurd, Iran
  4. Department of Biology, Faculty of Sciences, University of Sistan and Baluchestan, Zahedan, Iran
  5. Department of Medical Biotechnology and Cancer Gene Therapy Research Center, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
  6. Department of Virology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
  7. Fuller Laboratories, Fullerton, CA, USA
  8. Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT, USA

open access

Vol 58, No 3 (2020)
ORIGINAL PAPERS
Submitted: 2020-07-29
Accepted: 2020-09-08
Published online: 2020-09-16

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.

Get Citation

Keywords

CRISPR; Cas9; gRNA; genome editing; HSV-1; HEK-AD cells

About this article
Title

A knockdown of the herpes simplex virus type-1 gene in all-in-one CRISPR vectors

Journal

Folia Histochemica et Cytobiologica

Issue

Vol 58, No 3 (2020)

Article type

Original paper

Pages

174-181

Published online

2020-09-16

DOI

10.5603/FHC.a2020.0020

Pubmed

32937678

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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