RESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas , Genes Virales , Vectores Genéticos , Herpesvirus Humano 1/genética , Proteínas del Envoltorio Viral/genética , Proteína 9 Asociada a CRISPR/genética , Desoxirribonucleasa BamHI/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Técnicas de Silenciamiento del Gen , ARN Guía de Kinetoplastida , Programas InformáticosRESUMEN
This report explains the employing of a combination test of traditional cell culture with a quantitative real-time PCR for assessment of the antiviral effect of zinc sulfate (ZnSO4) on herpes simplex virus (HSV)-infected Vero cells. Our evidence showed that the treatment with 0.3 mM ZnSO4 strongly inhibited the replication of virus progeny (MOI 0.001) at least 68-fold less. On the other hand, the IC50 demonstrated that the highest activity of ZnSO4 was at the 0.23 mM concentration.