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Comparison of CRISPR-Cas9 tools for transcriptional repression and gene disruption in the BEVS
  • Mark Bruder,
  • Sadru-Dean Walji,
  • Marc Aucoin
Mark Bruder
University of Waterloo
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Sadru-Dean Walji
University of Waterloo
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Marc Aucoin
University of Waterloo
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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.