用CRISPR关联的转子子用于可编程的病毒载体工程和原始编辑
Quyen T Dang1, Chin-Wei Chang1, Pin-Yan Chen1
1Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300044, Taiwan.
Nucleic acids research
|February 23, 2026
概括
SHOT 2.0 是一种新的CRISPR相关的转子子平台,用于E. coli中的灵活的细菌工程. 这种工具可以在各种细胞类型中实现高效的基因传递和原始编辑,从而扩大了baculovirus的应用.
科学领域:
- 分子生物学分子生物学
- 基因编辑 基因编辑
- 病毒学 病毒学
背景情况:
- 百科病毒被广泛用于重组蛋白质表达和基因传递.
- 目前的 bacmid 工程方法缺乏灵活性和可编程性.
- 与CRISPR相关的转子子系统为先进的基因操纵提供了潜在的潜力.
研究的目的:
- 开发一个优化的CRISPR相关的转子子平台,用于灵活和可编程的细菌体工程.
- 为了使大型DNA货物能够有效地集成到特定的细菌体位置.
- 展示该平台用于构建用于基因编辑的先进的baculovirus载体的实用性.
主要方法:
- 开发SHOT 2.0,一种与CRISPR相关的转体子系统,用于在大肠杆菌中进行RNA引导的巴克米德编辑.
- 大型DNA有效载荷 (≥14kb) 的特定站点集成到bacmid位点 (v-cath,ODVe56).
- 构建和测试一个编码主要编辑器 (PE5max) 的全集baculovirus.
- 在HEK293T细胞,诱导多能干细胞 (iPSCs) 和肝癌细胞中评估主要编辑效率.
主要成果:
- 通过SHOT 2.0,可以将大型DNA货物 (≥14 kb) 具体集成到已定义的细菌体位置.
- 在ODVe56位点的整合显著提高了在连续病毒传递期间的转基因稳定性.
- 该系统兼容Bac-to-Bac®工作流,允许双基因插入.
- 矢量介导的原始编辑在HEK293T细胞中实现了高达85.6%的效率,在iPSC和肝癌细胞中达到37.1%.
结论:
- SHOT 2.0 显著扩展了 baculovirus 工程工具箱,为基因组编辑提供了一个灵活的平台.
- 该平台可为各种应用,包括基因传递,创建先进的细菌病毒载体.
- 在各种细胞类型中表现出强大的主要编辑能力,包括像iPSC和癌细胞这样具有挑战性的细胞.
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