Chakraborty Madhuja, Nielsen Lisa, Nash Delaney, Bruder Mark R, 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.
Int J Mol Sci. 2025 Jun 17;26(12):5805. doi: 10.3390/ijms26125805.
The Baculovirus Expression Vector System (BEVS) is an important protein and complex biologics production platform. The baculovirus GP64 protein is the major envelope glycoprotein that aids in virus entry and is required for cell-to-cell transmission in cell culture. Several studies have developed strategies around gene disruption in an attempt to minimize baculovirus co-production. Here, we investigate the result of transiently targeting the baculovirus gene with CRISPR-Cas9 during infection. Because not all genomes are effectively disrupted, we describe a variant calling methodology that allows the detection of the targeted mutations in even though these mutations are not the dominant sequences. Using a transfection-infection assay (T-I assay), the AcMNPV gene was targeted at six different locations to evaluate the effects of single and multiple targeting sites, and we demonstrated a reduction in the levels of baculovirus vectors while maintaining or enhancing foreign protein production when protein was driven by a p6.9 promoter. Viral genomes were subsequently isolated from the supernatant and cell pellet fractions, and our sequencing pipeline successfully detected indel mutations within for most of the single-guide RNA (sgRNA) targets. We also observed that 68.8% of variants found in the virus stock were conserved upon virus propagation in cell culture, thus indicating that they are not detrimental to viral fitness. This work provides a comprehensive assessment of CRISPR-Cas9 genome editing of baculovirus vectors, with potential applications in enhancing the efficiency of the BEVS.
杆状病毒表达载体系统（BEVS）是一个重要的蛋白质和复杂生物制品生产平台。杆状病毒GP64蛋白是主要的包膜糖蛋白，有助于病毒进入细胞，并且是细胞培养中细胞间传播所必需的。几项研究围绕基因破坏制定了策略，试图尽量减少杆状病毒的共产生。在这里，我们研究了在感染期间用CRISPR-Cas9瞬时靶向杆状病毒基因的结果。由于并非所有基因组都能被有效破坏，我们描述了一种变异检测方法，即使这些突变不是主要序列，也能检测到靶向突变。使用转染-感染试验（T-I试验），在六个不同位置靶向AcMNPV基因，以评估单个和多个靶向位点的效果，并且我们证明了当蛋白质由p6.9启动子驱动时，杆状病毒载体水平降低，同时维持或提高外源蛋白产量。随后从上清液和细胞沉淀部分分离病毒基因组，我们的测序流程成功检测到大多数单向导RNA（sgRNA）靶点内的插入缺失突变。我们还观察到，在细胞培养中病毒传代后，病毒原液中发现的68.8%的变异是保守的，因此表明它们对病毒适应性无害。这项工作对杆状病毒载体的CRISPR-Cas9基因组编辑进行了全面评估，在提高BEVS效率方面具有潜在应用价值。
