Rapid Generation of Recombinant Poxviruses Using CRISPR/Cas9 Gene Editing

Area of Science:

• Virology
• Molecular Biology
• Genetic Engineering

Background:

• Poxviruses, particularly vaccinia virus (VACV), are genetically modified using natural recombination for vaccine and cancer therapeutic applications.
• Traditional genetic modification methods are laborious, time-consuming, and often leave selectable markers.
• CRISPR/Cas9 technology has emerged as a tool to improve genetic manipulation of VACV.

Purpose of the Study:

• To develop an optimized method for rapid genetic engineering of VACV using CRISPR/Cas9.
• To introduce multiple genetic alterations in VACV efficiently and without selectable markers.
• To investigate the impact of CRISPR/Cas9 cut site positioning on recombination efficiency.

Main Methods:

• In vitro CRISPR/Cas9 genome editing of VACV DNA.
• Leporipoxvirus-mediated DNA repair and reactivation.
• Utilizing trans-provided repair fragments for introducing mutations and genetic alterations.
• Analysis of co-conversion of flanking markers based on CRISPR/Cas9 cut site.

Main Results:

• A streamlined method combining in vitro CRISPR/Cas9 editing and Leporipoxvirus-catalyzed repair was established.
• Multiple genetic modifications (substitutions, deletions, insertions) were introduced into VACV in a single step.
• The method eliminates the need for selectable markers, simplifying the generation of recombinant viruses.
• The positioning of CRISPR/Cas9-induced double-strand breaks influences the efficiency of marker co-conversion.

Conclusions:

• The developed method significantly accelerates the generation of genetically modified VACV.
• This approach facilitates the creation of recombinant VACV with multiple genetic alterations efficiently and without markers.
• The findings provide valuable insights for optimizing CRISPR/Cas9-based genome editing strategies in poxviruses.