Developing oncolytic Herpes simplex virus type 1 through UL39 knockout by CRISPR-Cas9.

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.