Department of Chemical Engineering, University of Waterloo, Waterloo, ON N2L 3G1, Canada.
Viruses. 2021 Sep 24;13(10):1925. doi: 10.3390/v13101925.
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 or 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.
利用 bacmid 的重组技术生成敲除病毒极大地加速了杆状病毒基因在各种应用中的研究。然而，与传统的重组和 RNAi 方法相比，CRISPR-Cas9 系统是一种简化基因的序列特异性基因组编辑和有效转录调控的强大工具。在这里，比较了 CRISPR-Cas9 系统在 BEVS 中用于基因敲除和转录抑制的效果。开发了持续表达 或 基因的细胞系，并评估了递送 sgRNA 的重组杆状病毒对报告绿色荧光蛋白基因的敲除或抑制效果。最后，针对内源性 AcMNPV 基因进行敲除或下调，以影响基因表达和杆状病毒复制。本研究提供了一个概念验证，表明 CRISPR-Cas9 技术可能是一种有效的工具，可通过靶向基因敲除和转录抑制来高效研究杆状病毒基因。
