A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors.

Area of Science:

• Molecular Biology
• Virology
• Gene Editing

Background:

• The CRISPR-associated endonuclease Cas9, guided by RNA, offers precise genomic modification capabilities.
• Previous applications of CRISPR/Cas9 have demonstrated success in editing the genomes of diverse organisms.

Purpose of the Study:

• To establish a protocol for editing the vaccinia virus (VV) genome within the cytoplasm of infected cells using the RNA-guided Cas9 system.
• To leverage CRISPR/Cas9-induced double-stranded DNA breaks for efficient homologous recombination and transgene integration in the VV genome.

Main Methods:

• Utilized the RNA-guided Cas9 system for targeted DNA modification in vaccinia virus (VV).
• Employed homologous recombination with a site-specific vector containing a transgene to insert genetic material into the targeted VV locus.
• Generated a modified VV by deleting the N1L gene and incorporating the red fluorescence protein (RFP) gene.

Main Results:

• Achieved efficient and specific double-stranded DNA breaks at the targeted VV genomic site.
• Demonstrated successful transgene incorporation into the VV genome via homologous recombination.
• Generated a N1L gene-deleted VV expressing RFP with a tenfold increase in recombination efficiency compared to conventional methods.

Conclusions:

• The RNA-guided Cas9 system provides a highly efficient method for generating mutant vaccinia viruses (VVs).
• This protocol enhances the efficiency of generating genetically modified VVs with integrated transgenes.