CRISPR/Cas9-mediated multiple single guide RNAs potently abrogate pseudorabies virus replication

Area of Science:

• Veterinary Virology
• Molecular Biology
• Gene Editing Technologies

Background:

• Pseudorabies virus (PRV) causes significant economic losses in the swine industry due to high morbidity and mortality.
• Current treatments for PRV infection are limited, and eradication from infected swine is challenging.

Purpose of the Study:

• To investigate the potential of the CRISPR/Cas9 system as a therapeutic strategy against PRV infections in swine.
• To identify effective single guide RNAs (sgRNAs) for targeting PRV genes.

Main Methods:

• Designed 75 sgRNAs targeting essential and non-essential genes of the PRV genome.
• Utilized a firefly luciferase-tagged reporter PRV virus for high-throughput screening of sgRNA efficacy.
• Employed transfection assays to evaluate the antiviral activity of sgRNAs, including combinations.

Main Results:

• A majority of the designed sgRNAs demonstrated significant inhibition of PRV replication.
• Simultaneous targeting of PRV with multiple sgRNAs completely abolished the production of infectious viruses in cell cultures.
• The CRISPR/Cas9 system shows potent antiviral activity against PRV.

Conclusions:

• CRISPR/Cas9 gene editing represents a promising novel therapeutic approach for controlling PRV infections.
• Targeting multiple PRV genes concurrently with sgRNAs offers a highly effective strategy to eliminate viral production.