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.

Anchorage-dependent cells are cells that require physical attachment to a solid surface, such as a culture dish, to survive, grow, and reproduce. In the biomedical industry, and others, having the ability to culture these cells is crucial, but current techniques used to separate cells from surfaces can induce stresses and reduce cell viability.

“In the pharmaceutical and biotechnology industries, cells are typically detached from culture surfaces using enzymes—a process fraught with challenges,” says Kripa Varanasi, MIT professor of mechanical engineering. “Enzymatic treatments can damage delicate cell membranes and surface proteins, particularly in primary cells, and often require multiple steps that make the workflow slow and labor-intensive.”

Existing approaches also rely on large volumes of consumables, generating an estimated 300 million liters of cell culture waste each year. Moreover, because these enzymes are often animal-derived, they can introduce compatibility concerns for cells intended for human therapies, limiting scalability and high-throughput applications in modern biomanufacturing.