question: INTRODUCTION Herpes simplex virus type 1 (HSV-1) 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), an important tool in genome engineering 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. MATERIAL 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. Also, 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 moreover, revealed 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 significant for the HSV1 infection and the development of new strategies for targeted therapy of viral infections caused by HSV-1.