Abstract
The generation of knock-out viruses using recombineering of bacmids has greatly accelerated scrutiny of baculovirus genes for a variety of applications. However, the CRISPR–Cas9 system is a powerful tool that simplifies sequence-specific genome editing and effective transcriptional regulation of genes compared to traditional recombineering and RNAi approaches. Here, the effectiveness of the CRISPR–Cas9 system for gene disruption and transcriptional repression in the BEVS was compared. Cell lines constitutively expressing the cas9 or dcas9 gene were developed, and recombinant baculoviruses delivering the sgRNA were evaluated for disruption or repression of a reporter green fluorescent protein gene. Finally, endogenous AcMNPV genes were targeted for disruption or downregulation to affect gene expression and baculovirus replication. This study provides a proof-of-concept that CRISPR–Cas9 technology may be an effective tool for efficient scrutiny of baculovirus genes through targeted gene disruption and transcriptional repression.
Highlights
Baculoviruses are a diverse group of enveloped viruses that contain large, doublestranded DNA genomes
Expression of the Cas9 and dCas9 proteins were conferred via the development of transgenic Sf9 cell lines transfected with plasmids pOpIE2-Cas9-puro and pOpIE2-dCas9puro, which include either the cas9 or dcas9 gene and the pac gene sequences separated by the viral T2A element [41]
After selection with puromycin for at least 2 weeks, resistant cells were pooled and maintained in suspension culture for an additional ~10–15 passages under selective pressure before removing the puromycin from the medium. Routine maintenance of these cell lines with or without puromycin provided no evidence that ectopic expression of either Cas9 protein had any effect on their growth, prior to performing any gene disruption or transcriptional repression experiments, the cell lines were characterized with infection experiments to determine whether there were any distinguishable differences between them and parental Sf9 cells
Summary
Baculoviruses are a diverse group of enveloped viruses that contain large, doublestranded DNA (dsDNA) genomes. A major milestone of BEVS biotechnology was the development of the bacmid system, which allowed site-specific integration of foreign genes in the AcMNPV genome propagated in E. coli [5]. Coupled with the λ-red or RecET recombineering systems [6,7], mutant AcMNPV genomes with sequence-specific deletions (i.e., knock-out viruses, KOVs) were efficiently obtained, allowing functional studies of many of the ~150 annotated open reading frames (ORFs) of AcMNPV [8,9,10,11,12,13]. Studies identifying per os infectivity factors, genes with insecticidal properties, and gene disruptions that improved recombinant protein production, have been conducted using bacmid technology [14,15,16,17]
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