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

Abstract

The alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) 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. AcMNPV 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 AcMNPV 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 AcMNPV gp64 gene disruption can minimize baculovirus contamination in cell cultures. Our findings provide a robust baseline for analyzing in process genome editing of baculoviruses.

Keywords: AcMNPV; CRISPR-Cas9; baculovirus; bioinformatics pipeline; indel mutation; next-generation sequencing; transfection-infection assay.

Figure 1

Odd-numbered forward (L) and reverse (R) primers are combined to make Primer Pool 1 and even-numbered forward (L) and reverse (R) primers are combined to make Primer Pool 2.

Figure 2

Parameters used for the four Trimmomatic operations. The * here indicates a mandatory field.

Figure 3

Impact of sgRNA rBEV-mediated gp64 gene disruption on (a) foreign protein production and (b) infectious virus titer. The data from 3 replicates are presented here.

Figure 4

Effect on (a) foreign protein production and (b) infectious virus titer upon CRISPR-Cas9 based T-I assay. The data from three replicates are presented here.