Exploring Poxvirus Genome Packaging using CRISPR-Cas Systems

The vaccinia virus genome is an approximately 195-kb linear double-stranded DNA genome that is AT-rich (~67%) and covalently closed at both ends. How this genome is replicated and packaged is still being determined. Many viruses rely on genomic elements called packaging signals to selectively encapsidate viral DNA/RNA over cellular DNA/RNA. To identify the location of packaging signals, the vaccinia virus genome can be targeted by CRISPR-Cas endonucleases that cleave DNA preceded by a short PAM. The CRISPR-Cas system can fragment the vaccinia virus genome and, with the help of fluorescent protein reporters, determine packaged fragments, if there are any. The process of developing a FnCas12a system and SpCas9 system that targets the vaccinia virus genome is described in this paper.