Nastaran Khodadad1,2, Mona Fani1,3, Saleh Jamehdor4, Rahil Nahidsamiei1,2, Manoochehr Makvandi1,2, Saeed Kaboli5, Ali Teimoori6,7,8, Jose Thekkiniath9,10. 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. Cancer Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. teimooriali1982@gmail.com. 7. Department of Virology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. teimooriali1982@gmail.com. 8. Department of Virology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran. teimooriali1982@gmail.com. 9. Fuller Laboratories, Fullerton, CA, USA. 10. Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT, USA.
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.
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.