使用单蛋白CRISPR效应器Cas7-11进行可编程RNA向
Ahsen Özcan1, Rohan Krajeski1, Eleonora Ioannidi1,2
1McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA.
Nature
|September 7, 2021
概括
研究人员发现了一种新型单蛋白CRISPR-Cas效应器Cas7-11. 这种Cas7-11系统有效地以最小的毒性准RNA, 为RNA干扰和编辑提供了一个新的工具.
科学领域:
- 分子生物学
- 遗传学
- 生物技术
背景情况:
- 克里斯普尔-卡斯干扰依赖于卡斯核酶,在多子单元 (类1) 或单蛋白 (类2) 系统中发现.
- 了解CRISPR-Cas效应器的多样性和演变对于开发新的生物技术工具至关重要.
研究的目的:
- 描述1类CRISPR-Cas系统中的Cas7-11效应器.
- 评估其作为可编程RNA向基因编辑和淘汰工具的潜力.
- 与现有的RNA向技术相比,评估其安全性和有效性.
主要方法:
- 在大肠杆菌中表达和净化Desulfonema ishimotonii Cas7-11 (DiCas7-11).
- 在体外鉴定DiCas7-11RNA干扰活性,包括crRNA前处理和点RNA分裂.
- 在哺乳动物细胞中用于RNA敲除和编辑的DiCas7-11工程.
- 与shRNA和Cas13相比,DiCas7-11的细胞活力和毒性的评估
主要成果:
- 在Cas11和Cas7域的融合中,DiCas7-11作为1类CRISPR-Cas系统中的单蛋白效应体.
- DiCas7-11对mRNA和细菌菌具有强烈的RNA干扰,处理crRNA和分离目标RNA.
- 在哺乳动物细胞中,设计的 DiCas7-11 证明了有效的 RNA 淘汰和编辑,但对细胞活力没有观察到影响.
- 与短毛针RNA和Cas13相比,Cas7-11的细胞毒性显著降低.
结论:
- Cas7-11 是一种进化中间体,从多个子单元的第1类效应器过渡到单一蛋白质的第2类效应器.
- 这项研究扩大了已知的CRISPR-Cas系统和效应机制的多样性.
- Cas7-11是一种有前途的,无毒的,可编程的RNA向工具,用于各种生物技术应用,包括基因编辑和RNA干扰.
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