Rapid poxvirus engineering using CRISPR/Cas9 as a selection tool

Area of Science:

• Molecular Biology
• Virology
• Gene Editing

Background:

• CRISPR/Cas9 technology typically relies on efficient DNA repair following genome cutting for genetic modification.
• Engineering large dsDNA viruses like poxviruses using standard CRISPR/Cas9 methods has been challenging due to repair inefficiencies.

Purpose of the Study:

• To investigate the efficiency of CRISPR/Cas9-mediated DNA repair in poxviruses.
• To explore alternative applications of CRISPR/Cas9 in poxvirus genome engineering.

Main Methods:

• Utilized CRISPR/Cas9 (Cas9 and guide RNA) complexes to target poxvirus genomes post-entry into host cells.
• Assessed homologous recombination rates between repair constructs and viral genomes.
• Monitored poxvirus DNA replication and viral spread in cell culture.

Main Results:

• Cas9-guide RNA complexes effectively cleaved poxvirus genomes but resulted in inefficient repair of the induced breaks.
• Cas9 cleavage modestly improved, but did not significantly enhance, homologous recombination rates.
• CRISPR/Cas9 targeting unexpectedly inhibited poxvirus DNA replication and suppressed virus spread.

Conclusions:

• CRISPR/Cas9 acts as an antiviral mechanism in poxviruses by inhibiting replication, rather than solely facilitating precise genome editing.
• This inhibitory effect provides a novel method for selecting conventionally generated poxvirus recombinants without marker genes.
• The application of CRISPR/Cas9 significantly accelerates the generation of poxvirus-based vaccines, enhancing their utility for personalized cancer therapies and rapid response to emerging diseases.