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Developing oncolytic Herpes simplex virus type 1 through UL39 knockout by CRISPR-Cas9
Saeedeh Ebrahimi1, Manoochehr Makvandi1, Samaneh Abbasi2
1Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Objectives:
Oncolytic Herpes simplex virus type 1 (HSV-1) has emerged as a promising strategy for cancer therapy. However, development of novel oncolytic mutants has remained a major challenge owing to low efficiency of conventional genome editing methods. Recently, CRISPR-Cas9 has revolutionized genome editing.
Materials And Methods:
In this study, we aimed to evaluate the capability of CRISPR-Cas9 to manipulate the UL39 gene to create oncolytic HSV-1. Herein, three sgRNAs were designed against the UL39 gene and transfected into HEK-293 cell line followed by infection with HSV-1 KOS.
Results:
After three rounds of plaque purification, several HSV-1 mutants were identified by PCR analysis and sequencing. One of these mutations in which 55 nucleotides were deleted resulted in a frameshift mutation that in turn produced a truncated protein with only 167 amino acids from 1137 amino acids. Functional analysis in Vero and primary fibroblast cells revealed that viral replication was significantly lower and plaque size was smaller in the HSV-1 mutant compared with HSV-1 KOS. Moreover, the relative amount of viral genome present in the supernatants of infected cells (Vero and primary fibroblast cells) with HSV-1 mutant was significantly decreased compared with those of HSV-1 KOS.
Conclusion:
Our data revealed that targeting UL39 with CRISPR-Cas9 could develop oncolytic HSV-1.
Insights
CRISPR-Cas9 gene editing effectively created oncolytic Herpes simplex virus type 1 (HSV-1) by targeting the UL39 gene. This novel oncolytic HSV-1 mutant demonstrated reduced viral replication and genome presence, showing promise for cancer therapy.
Area of Science:
- Virology
- Cancer Therapy
- Gene Editing
Background:
- Oncolytic Herpes simplex virus type 1 (HSV-1) shows potential for cancer treatment.
- Developing novel oncolytic HSV-1 mutants is challenging due to inefficient conventional genome editing.
- CRISPR-Cas9 technology offers a revolutionary approach to genome editing.
Purpose of the Study:
- To assess CRISPR-Cas9's efficacy in modifying the UL39 gene of HSV-1 to generate oncolytic HSV-1.
- To create and characterize a novel oncolytic HSV-1 mutant using CRISPR-Cas9.
Main Methods:
- Designed three single-guide RNAs (sgRNAs) targeting the UL39 gene.
- Transfected sgRNAs into HEK-293 cells, followed by HSV-1 KOS infection.
- Identified and purified HSV-1 mutants using PCR and sequencing.
Main Results:
- A 55-nucleotide deletion in UL39 caused a frameshift mutation, producing a truncated protein.
- The HSV-1 mutant exhibited significantly reduced viral replication and smaller plaque sizes compared to HSV-1 KOS.
- A decreased relative amount of viral genome was observed in supernatants of cells infected with the HSV-1 mutant.
Conclusions:
- Targeting the UL39 gene with CRISPR-Cas9 is a viable strategy for developing oncolytic HSV-1.
- The generated oncolytic HSV-1 mutant shows potential for enhanced cancer therapy applications.
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