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The Sf9 cell line, originally isolated from the insect Spodoptera frugiperda, is commonly used alongside the baculovirus expression vector system (BEVS) to produce recombinant proteins and other biologics. As more BEVS-derived vaccines and therapeutics are approved by regulators and manufactured at scale, there is increasing interest in improving the Sf9 cell line to improve bioprocess robustness and increase product yields. CRISPR-Cas9 is a powerful genome-editing tool with great potential to improve cell line characteristics. Nevertheless, reports of genome-editing in Sf9 cells are scarce, and targets for engineering are elusive. To evaluate the effectiveness of CRISPR-Cas9 to improve BEVS yields, we generated Sf9 cell lines with functional knockouts in the Sf-Caspase-1 gene, which encodes an effector caspase involved in the execution of apoptosis. Deletion of Sf-Caspase-1 abolished the hallmarks of apoptotic cell death including plasma membrane blebbing and effector caspase activity. Following infection of Sf-Caspase-1 knockout Sf9 cultures with a recombinant baculovirus expressing βgalactosidase, we did not observe any differences in cell death kinetics or increases in productivity. Similar results were obtained when Sf-Caspase-1 expression was suppressed via RNA interference. We anticipate that the CRISPR-Cas9 workflow reported here will spur future efforts to rationally engineer Sf9 cells for improved baculovirus expression.

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The Sf9 cell line, originally isolated from the ovarian tissue of Spodoptera frugiperda larvae, is widely used in academia and industry for the baculovirus-mediated production of recombinant proteins and virus-like particles. RNA interference (RNAi) is a conserved antiviral pathway present in eukaryotic organisms and is the primary antiviral defence mechanism in insects. Recent evidence has implicated RNAi as an antiviral response to baculovirus infection in Sf9 cells. To test this hypothesis, CRISPR/Cas9 technology was used to disable the RNAi pathway in Sf9 cells by knocking out Dicer-2, the protein responsible for cleaving viral double-stranded RNA precursors into short interfering RNAs. Infection of Dicer-2 knockout Sf9 cells with either the wild-type baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV), recombinant AcMNPV (rAcMNPV) expressing β-galactosidase (β-gal), or rAcMNPV expressing a wasp venom protein (Vn50) at a multiplicity of infection (m.o.i.) of 1 resulted in a modest increase in virus replication compared to control Sf9 cells under adherent culture conditions. In contrast, Dicer-2 knockout Sf9 monolayer or suspension cultures infected by the rAcMNPV expressing β-gal at higher m.o.i.s (3.5 and 20) did not exhibit increases in either viral DNA replication or β-gal production. Intriguingly, during long-term passaging in suspension, Dicer-2 knockout Sf9 cultures underwent transient crashes in cell proliferation and viability. It was discovered that these periods of low growth and viability coincided with a dramatic increase in the RNA levels of S. frugiperda rhabdovirus, a recently identified adventitious virus that persistently infects the Sf9 cell line, suggesting a role for Dicer-2 in managing chronic viral infections in this industrially relevant insect cell line.