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使用双重gRNA优化重组蛋白质生产的AcMNPV的CRISPR/Cas9驱动工程

Rocco Valente^1,2, Joaquín Poodts^1,2, Joaquín Manuel Birenbaum^1,2

¹Cátedra de Biotecnología, Departamento de Microbiología, Inmunología, Biotecnología y Genética, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, Buenos Aires C1113AAD, Argentina.

|August 28, 2025

概括

这项研究引入了双向导CRISPR/Cas9用于baculovirus基因组工程,创建删除以增强昆虫细胞和幼虫的重组蛋白质生产. 这种方法有效地去除不必要的基因,导致蛋白质产量有所增加,但有时是显著的.

科学领域:

分子生物学
基因组学
生物技术

背景情况:

CRISPR/Cas9系统是一个多功能基因组编辑工具.
百科病毒表达载体系统 (BEVS) 广泛用于重组蛋白质的生产.
优化细菌病毒基因组可以提高蛋白质产量.

研究的目的:

报告首次使用双单导向RNA (sgRNA) CRISPR/Cas9用于Autographa californica多重核聚合体病毒 (AcMNPV) 基因组删除.
评估这些缺失对生物生产中的重组蛋白质表达的影响.
验证CRISPR/Cas9作为工程最小化细菌病毒基因组的工具.

主要方法:

在AcMNPV中使用双 sgRNA CRISPR/Cas9方法进行定向基因组删除.
同时向Sf9昆虫细胞输送两个不同的核糖蛋白 (RNP) 复合体.
在baculovirus基因组中生成基因片段的删除,包括合基因.
在昆虫细胞和幼虫中评估重组蛋白 (eGFP和HRPc) 的表达.

主要成果:

使用双导向CRISPR/Cas9系统有效生成AcMNPV基因组删除.
基因缺失对重组蛋白质表达有多种影响,其中一些增强了产量.
值得注意的是,HRPc表达在Spodoptera frugiperda幼虫中增加了多达3.1倍.
根据宿主系统,证明非必需基因去除会对蛋白质产量产生不同的影响.

结论:

双向导CRISPR/Cas9编辑是一种快速而精确的细菌病毒基因组工程方法.
这一策略可以开发最小化的细菌病毒基因组,以改善生物生产.
基因删除的宿主依赖性影响突出了优化BEVS的复杂性.

关键词:

美国国务院在CRISPR/Cas9中拉奇普鲁西亚现在在 Sf9 细胞中类动物细菌病毒双 sgRNA 的时间表达基因组编辑淘汰赛优化情况

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