Evaluation of Virus-Free Manufacture of Recombinant Proteins Using CRISPR-Mediated Gene Disruption in Baculovirus-Infected Insect Cells.

The manufacture and downstream processing of virus-like particles (VLPs) using the baculovirus expression vector system (BEVS) is complicated by the presence of large concentrations of baculovirus particles, which are similar in size and density to VLPs, and consequently are difficult to separate. To reduce the burden of downstream processing, CRISPR-Cas9 technology was used to introduce insertion-deletion (indel) mutations within the multiple nucleopolyhedrovirus (MNPV) open reading frame, which encodes the major envelope protein of MNPV. After comfirming the site-specific targeting of leading to reduced budded virus (BV) release, the gene of human immunodeficiency virus type 1 was expressed to produce Gag VLPs. This approach was effective for producing VLPs using the BEVS whilst simultaneously obstructing BV release.