Synthetic biology applied to baculoviruses enables genome optimization through the targeted deletion of nonessential genes, enhancing recombinant protein expression. In this study, the CRISPR/Cas9 system with two tandem sgRNAs was used and validated as an efficient strategy to remove independently large genomic fragments from AcMNPV, all encoding proteins dispensable for budded virus production. The resulting mutant viruses were evaluated for their ability to express eGFP and HRPc in Sf9 cells and in Rachiplusia nu and Spodoptera frugiperda larvae. Deletions of the Ac15–Ac16 and Ac129–Ac131 regions led to significant increases in protein expression in infected cells and in larvae. In contrast, deletion of the Ac136–Ac138 region enhanced expression only in cultured cells but had a negative impact on larvae. Removal of the Ac148–Ac150 fragment caused a marked reduction in expression in both experimental systems. These findings confirm that although some genes are nonessential for systemic infection, their combined deletion can differentially affect recombinant protein expression depending on the host. This study validates not only an effective strategy for developing minimized baculovirus genomes but also the use of dual-guide CRISPR/Cas9 editing as a rapid and precise tool for genome engineering in baculovirus-based expression systems.

The CRISPR/Cas9 system is a powerful genome-editing tool that is applied in baculovirus engineering. In this study, we present the first report of the AcMNPV genome deletions for bioproduction purposes, using a dual single-guide RNA (sgRNA) CRISPR/Cas9 approach. We used this method to remove nonessential genes for the budded virus and boost recombinant protein yields when applied as BEVS. We show that the co-delivery of two distinct ribonucleoprotein (RNP) complexes, each assembled with a sgRNA and Cas9, into Sf9 insect cells efficiently generated deletions of fragments containing tandem genes in the genome. To evaluate the potential of this method, we assessed the expression of two model proteins, eGFP and HRPc, in insect cells and larvae. The gene deletions had diverse effects on protein expression: some significantly enhanced it while others reduced production. These results indicate that, although the targeted genes are nonessential, their removal can differentially affect recombinant protein yields depending on the host. Notably, HRPC expression increased up to 3.1-fold in Spodoptera frugiperda larvae. These findings validate an effective strategy for developing minimized baculovirus genomes and demonstrate that dual-guide CRISPR/Cas9 editing is a rapid and precise tool for baculovirus genome engineering.