Chakraborty Madhuja, Nielsen Lisa, Nash Delaney, Nissimov Jozef I, Charles Trevor C, Aucoin Marc G
Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada.
Department of Biology, University of Waterloo, Waterloo, ON N2L 3G1, Canada.
Viruses. 2025 Mar 18;17(3):437. doi: 10.3390/v17030437.
The alphabaculovirus multiple nucleopolyhedrovirus (MNPV) is the most commonly used virus in the Baculovirus Expression Vector System (BEVS) and has been utilized for the production of many human and veterinary biologics. MNPV has a large dsDNA genome that remains understudied, and relatively unmodified from the wild-type, especially considering how extensively utilized it is as an expression vector. Previously, our group utilized CRISPR-Cas9 genome engineering that revealed phenotypic changes when baculovirus genes are targeted using either co-expressed sgRNA or transfected sgRNA into a stable insect cell line that produced the Cas9 protein. Here, we describe a pipeline to sequence the recombinant MNPV expression vectors using shotgun sequencing, provide a set of primers for tiled-amplicon sequencing, show that untargeted baculovirus vector genomes remain relatively unchanged when amplified in Sf9-Cas9 cells, and confirm that MNPV gene disruption can minimize baculovirus contamination in cell cultures. Our findings provide a robust baseline for analyzing genome editing of baculoviruses.
甲型杆状病毒多角体核型多角体病毒（MNPV）是杆状病毒表达载体系统（BEVS）中最常用的病毒，已被用于生产多种人用和兽用生物制品。MNPV具有一个大型双链DNA基因组，该基因组仍未得到充分研究，并且与野生型相比相对未被修饰，特别是考虑到它作为表达载体被广泛使用的程度。此前，我们的研究小组利用CRISPR-Cas9基因组工程技术发现，当使用共表达的sgRNA或将sgRNA转染到产生Cas9蛋白的稳定昆虫细胞系中靶向杆状病毒基因时，会出现表型变化。在此，我们描述了一种使用鸟枪法测序对重组MNPV表达载体进行测序的流程，提供了一组用于平铺扩增子测序的引物，表明在Sf9-Cas9细胞中扩增时，未靶向的杆状病毒载体基因组保持相对不变，并证实MNPV基因破坏可将细胞培养物中的杆状病毒污染降至最低。我们的研究结果为分析杆状病毒的基因组编辑提供了一个可靠的基线。
