类型III-E CRISPR-Cas7-11效应器组合的结构和工程
Kazuki Kato1, Wenyuan Zhou2, Sae Okazaki1
1Structural Biology Division, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
Cell
|June 1, 2022
概括
Cas7-11 CRISPR-Cas系统为细菌和哺乳动物细胞提供双重RNA向能力. 结构洞察力使得能够设计一个紧的Cas7-11变体,用于使用AAV载体的体内RNA倒置应用.
科学领域:
- 分子生物学
- 结构生物学
- 基因编辑技术
背景情况:
- 类型III-E CRISPR-Cas效应器,Cas7-11,具有独特的双RNase活动.
- 这些活动包括前体CRISPRRNA (前crRNA) 处理和CRISPRRNA (crRNA) 引导的点RNA分裂.
- 在细菌和哺乳动物系统中,Cas7-11代表了RNA向的新平台.
研究的目的:
- 确定与crRNA和点RNA结合的Cas7-11复合物的高分辨率结构.
- 阐明Cas7-11双重RNase活动的分子机制.
- 为体内应用设计一个紧的Cas7-11变种.
主要方法:
- 低温电子显微镜 (cryo-EM) 在2.5-Å分辨率.
- 生物化学测试以确认催化活性.
- 在Cas7-11的合理蛋白质工程.
主要成果:
- 这项研究揭示了Cas7-11的模块化架构,包括七个域和四个链接器.
- 结构分析确定了特定域在crRNA处理和目标RNA识别中的作用.
- 生物化学数据与有关RNA裂变的催化残留物定位的结构发现相关.
- 一个紧的Cas7-11变种 (Cas7-11S) 成功设计.
结论:
- 确定的结构为Cas7-11针对RNA的作用机制提供了关键的见解.
- 设计的Cas7-11S促进了单向量AAV包装,以有效地破坏人类细胞中的转录.
- 这项工作使Cas7-11系统在RNA调制中的未来应用成为可能.
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