黄金葡萄球菌Cas9的晶体结构
Hiroshi Nishimasu1, Le Cong2, Winston X Yan3
1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; JST, PRESTO, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Cell
|August 29, 2015
概括
研究人员阐明了Staphylococcus aureus Cas9 (SaCas9) 的晶体结构,揭示了它识别多种DNA点的机制. 这种结构洞察力可以开发先进的CRISPR-Cas9基因组编辑工具.
科学领域:
- 生物化学
- 分子生物学
- 结构生物学
背景情况:
- CRISPR-Cas9技术依赖于像Cas9这样的RNA导向DNA内核酶进行基因组编辑.
- 黄金葡萄球菌Cas9 (SaCas9) 是一种较小,更易于管理的替代体,用于体内应用.
研究的目的:
- 确定与指导RNA和向DNA结合的SaCas9的高分辨率晶体结构.
- 阐明 SaCas9 宽松原空间器相邻动机 (PAM) 识别的结构基础.
- 将SaCas9和SpCas9结构进行比较,以了解PAM特异性和指导RNA结合的差异.
主要方法:
- 使用X射线结晶学来获得SaCas9-sgRNA-DNA复合物的结构.
- 进行了SaCas9和SpCas9之间的结构分析和比较.
主要成果:
- 在2. 6和2. 7 Å分辨率下确定了SaCas9具有两个不同的DNA点 (5'-TTGAAT-3' PAM和5'-TTGGGT-3' PAM) 的晶体结构.
- 这些结构揭示了SaCas9对5'-NNGRRT-3' PAM序列的放松识别机制.
- 结构性比较突出了SaCas9和SpCas9之间的保留和分歧特征,解释了它们不同的PAM特点.
结论:
- 对SaCas9的结构洞察力为了解其独特的PAM认可提供了基础.
- 这些知识有助于合理设计基于CRISPR-Cas9的新型基因组编辑工具,包括转录激活剂和可诱导核酶.
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