CRISPR/Cas9-Driven Engineering of AcMNPV Using Dual gRNA for Optimized Recombinant Protein Production

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• The CRISPR/Cas9 system is a versatile genome-editing tool.
• Baculovirus expression vector systems (BEVS) are widely used for recombinant protein production.
• Optimizing baculovirus genomes can enhance protein yields.

Purpose of the Study:

• To report the first application of dual single-guide RNA (sgRNA) CRISPR/Cas9 for Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome deletions.
• To assess the impact of these deletions on recombinant protein expression for bioproduction.
• To validate CRISPR/Cas9 as a tool for engineering minimized baculovirus genomes.

Main Methods:

• Utilized a dual sgRNA CRISPR/Cas9 approach for targeted genome deletions in AcMNPV.
• Co-delivered two distinct ribonucleoprotein (RNP) complexes into Sf9 insect cells.
• Generated deletions of gene fragments, including tandem genes, within the baculovirus genome.
• Evaluated recombinant protein (eGFP and HRPc) expression in insect cells and larvae.

Main Results:

• Efficient generation of AcMNPV genome deletions using the dual-guide CRISPR/Cas9 system.
• Gene deletions exhibited varied effects on recombinant protein expression, with some enhancing yields.
• Notably, HRPc expression increased up to 3.1-fold in *Spodoptera frugiperda* larvae.
• Demonstrated that nonessential gene removal impacts protein yield differently based on the host system.

Conclusions:

• Dual-guide CRISPR/Cas9 editing is a rapid and precise method for baculovirus genome engineering.
• This strategy enables the development of minimized baculovirus genomes for improved bioproduction.
• Host-dependent effects of gene deletions highlight the complexity of optimizing BEVS.