Department of Chemical Engineering, University of Waterloo, 200 University Ave. W., Waterloo, N2L 3G1, Ontario, Canada.
Appl Microbiol Biotechnol. 2023 Jul;107(13):4323-4335. doi: 10.1007/s00253-023-12462-y. Epub 2023 May 26.
Baculoviruses have very large genomes and previous studies have demonstrated improvements in recombinant protein production and genome stability through the removal of some nonessential sequences. However, recombinant baculovirus expression vectors (rBEVs) in widespread use remain virtually unmodified. Traditional approaches for generating knockout viruses (KOVs) require several experimental steps to remove the target gene prior to the generation of the virus. In order to optimize rBEV genomes by removing nonessential sequences, more efficient techniques for establishing and evaluating KOVs are required. Here, we have developed a sensitive assay utilizing CRISPR-Cas9-mediated gene targeting to examine the phenotypic impact of disruption of endogenous Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genes. For validation, 13 AcMNPV genes were targeted for disruption and evaluated for the production of GFP and progeny virus - traits that are essential for their use as vectors for recombinant protein production. The assay involves transfection of sgRNA into a Cas9-expressing Sf9 cell line followed by infection with a baculovirus vector carrying the gfp gene under the p10 or p6.9 promoters. This assay represents an efficient strategy for scrutinizing AcMNPV gene function through targeted disruption, and represents a valuable tool for developing an optimized rBEV genome. KEY POINTS: [Formula: see text] A method to scrutinize the essentiality of baculovirus genes was developed. [Formula: see text] The method uses Sf9-Cas9 cells, a targeting plasmid carrying a sgRNA, and a rBEV-GFP. [Formula: see text] The method allows scrutiny by only needing to modify the targeting sgRNA plasmid.
杆状病毒具有非常大的基因组，先前的研究表明，通过去除一些非必需序列，可以提高重组蛋白的生产和基因组的稳定性。然而，广泛使用的重组杆状病毒表达载体 (rBEV) 实际上仍然没有经过修改。生成敲除病毒 (KOV) 的传统方法需要经过几个实验步骤，在生成病毒之前需要去除靶基因。为了通过去除非必需序列来优化 rBEV 基因组，需要更有效的技术来建立和评估 KOV。在这里，我们开发了一种利用 CRISPR-Cas9 介导的基因靶向的敏感测定法，以检查破坏内源性加利福尼亚多角体病毒 (AcMNPV) 基因对表型的影响。为了验证，我们针对 13 个 AcMNPV 基因进行了靶向破坏，并评估了 GFP 的产生和后代病毒 - 这些特性对于它们作为重组蛋白生产载体的使用是必不可少的。该测定法涉及将 sgRNA 转染到 Cas9 表达的 Sf9 细胞系中，然后用携带 GFP 基因的杆状病毒载体感染，该载体在 p10 或 p6.9 启动子下。该测定法代表了通过靶向破坏仔细研究 AcMNPV 基因功能的有效策略，并且是开发优化的 rBEV 基因组的有价值的工具。要点：[公式：见文本] 开发了一种用于仔细研究杆状病毒基因的必需性的方法。[公式：见文本] 该方法使用 Sf9-Cas9 细胞、携带 sgRNA 的靶向质粒和 rBEV-GFP。[公式：见文本] 该方法仅通过修改靶向 sgRNA 质粒即可进行检查。
