通过CRISPRRNA引导的内核酶Cas9进行DNA审讯
Samuel H Sternberg1, Sy Redding2, Martin Jinek3
11] Department of Chemistry, University of California, Berkeley, California 94720, USA [2].
Nature
|January 31, 2014
概括
对于基因组工程至关重要的Cas9酶,需要一种特定的DNA序列,称为原空间体相邻基因 (PAM),用于结合和切割. PAM的承认决定了Cas9的情况.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
- 遗传学 是一个遗传学.
- 基因组工程是基因组工程.
背景情况:
- 聚合定期间隔的短平行体重复 (CRISPR) 相关酶Cas9是一种强大的RNA引导的内核酶.
- Cas9指导RNA复合体被广泛用于各种生物体的基因组工程.
- 了解Cas9 DNA查询的精确机制对于其有效应用至关重要.
研究的目的:
- 阐明Cas9-RNA复合体与DNA相互作用的分子机制.
- 为了确定Cas9如何识别特定的DNA裂变部位.
- 为了研究原体邻基因 (PAM) 在Cas9结合和活性中的作用.
主要方法:
- 单分子生物化学实验. 单分子生物化学实验.
- 大量生物化学测试.
- 竞争试验用于研究DNA结合和链分离动态.
主要成果:
- Cas9-RNA的结合和DNA裂变取决于对三核酸原空间器相邻动机 (PAM) 的识别.
- DNA 结合亲和力与 PAM 密度相关;缺乏 PAM 的序列不会被准,即使它们与导向 RNA 互补.
- PAM识别启动了DNA链分离和RNA-DNA异重复组的形成,以定向进行,并触发了Cas9的催化活性.
结论:
- Cas9利用PAM识别作为快速扫描大型DNA分子和识别潜在目标站点的关键步骤.
- 该PAM序列作为一个调节元件,控制由Cas9.9启动DNA裂变.
- 这些发现为Cas9介导的基因组编辑的特异性和调节提供了基本的见解.
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