在大肠杆菌中寻找dCas9的动力学
Daniel Lawson Jones1, Prune Leroy1, Cecilia Unoson1
1Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
概括
一个Cas9蛋白需要6个小时才能在大肠杆菌中找到它的目标DNA序列. 高度的Cas9和指导RNA是快速向DNA所必需的.
科学领域:
- 分子生物学
- 遗传学
- 微生物学
背景情况:
- 这种由RNA指导的Cas9蛋白可以准特定的DNA序列.
- 这个过程需要DNA解来验证基因配对.
- 了解这种搜索速度对于基因编辑应用至关重要.
研究的目的:
- 确定Cas9蛋白在活细胞中定位特定染色体DNA序列所需的时间.
- 研究催化不活性Cas9 (dCas9) 的细胞内搜索机制.
主要方法:
- 使用单分子光显微镜.
- 使用的批量限制保护试验.
- 在活体大肠杆菌中研究dCas9.
主要成果:
- 一个被光标记的dCas9蛋白需要6个小时才能找到它的目标DNA序列.
- 每个潜在的目标DNA序列的结合时间不到30毫秒.
- 一旦结合,dCas9将与DNA保持关联,直到复制.
- 高度的Cas9和指导RNA对于快速向至关重要.
结论:
- Cas9对特定DNA序列的细胞内搜索是一个缓慢的过程.
- 通过Cas9有效和快速的DNA向需要蛋白质及其指导RNA的高细胞内度.
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