Comparison of CRISPR-Cas9 tools for transcriptional repression and gene
disruption in the BEVS
Abstract
The baculovirus expression vector system (BEVS) is a robust and
customizable platform for producing recombinant proteins for basic
research and biomedical applications. However, genome instability is an
intrinsic property of BEVs, and expression of several viral proteins
negatively impacts recombinant protein quantity and quality. The
CRISPR-Cas9 system is a powerful tool that simplifies sequence-specific
genome editing and effective transcriptional regulation of genes for
which disruption may not be appropriate. Here, the effectiveness of the
CRISPR-Cas9 system for gene disruption and transcriptional repression in
the BEVS was compared. A cell line constitutively expressing the cas9 or
dcas9 gene was developed, and recombinant baculoviruses delivering the
sgRNA were evaluated for disruption or repression of a reporter gfp
gene. Finally, endogenous AcMNPV genes were targeted for disruption or
downregulation to affect gene expression and baculovirus replication.
This development lays a foundation for optimization of the BEV for
improved genome stability and recombinant protein production.