Adapting Next-Generation Sequencing to CRISPR-Cas9 Genome Editing of Recombinant MNPV Vectors: From Shotgun to Tiled-Amplicon Sequencing.

The alphabaculovirus multiple nucleopolyhedrovirus (MNPV) is the most commonly used virus in the Baculovirus Expression Vector System (BEVS) and has been utilized for the production of many human and veterinary biologics. MNPV has a large dsDNA genome that remains understudied, and relatively unmodified from the wild-type, especially considering how extensively utilized it is as an expression vector. Previously, our group utilized CRISPR-Cas9 genome engineering that revealed phenotypic changes when baculovirus genes are targeted using either co-expressed sgRNA or transfected sgRNA into a stable insect cell line that produced the Cas9 protein. Here, we describe a pipeline to sequence the recombinant MNPV expression vectors using shotgun sequencing, provide a set of primers for tiled-amplicon sequencing, show that untargeted baculovirus vector genomes remain relatively unchanged when amplified in Sf9-Cas9 cells, and confirm that MNPV gene disruption can minimize baculovirus contamination in cell cultures. Our findings provide a robust baseline for analyzing genome editing of baculoviruses.