Adapting Next-Generation Sequencing to in Process CRISPR-Cas9 Genome Editing of Recombinant AcMNPV Vectors: From Shotgun to Tiled-Amplicon Sequencing

The alphabaculovirus <i>Autographa californica</i> multiple nucleopolyhedrovirus (<i>Ac</i>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. <i>Ac</i>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 <i>Ac</i>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 <i>Ac</i>MNPV <i>gp64</i> gene disruption can minimize baculovirus contamination in cell cultures. Our findings provide a robust baseline for analyzing <i>in process</i> genome editing of baculoviruses.