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Updated: Mar 25, 2026
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Genome Editing in Mammalian Cell Lines using CRISPR-Cas
Published on: April 11, 2019
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弗朗西塞拉·诺维西达的结构和工程
Hisato Hirano1, Jonathan S Gootenberg2, Takuro Horii3
1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Cell
|February 16, 2016
概括
研究人员描述了Francisella novicida Cas9 (FnCas9) 的结构,揭示了CRISPR-Cas9系统中保存和分离的特征. 他们设计了一种能够识别更广泛的PAM序列的变体, 扩大了基因组编辑能力.
科学领域:
- 结构生物学
- 分子生物学
- 基因组工程
背景情况:
- 在RNA的指导下,Cas9酶进行双链DNA分裂以进行可编程的基因组编辑.
- Cas9活动受到特定原空间器相邻基因 (PAM) 序列的限制.
- 了解Cas9-PAM相互作用对于扩展其基因组编辑应用至关重要.
研究的目的:
- 确定与指导RNA和DNA点复合的Francisella novicida Cas9的高分辨率晶体结构.
- 将FnCas9结构与其他Cas9正态相比较,以确定保存和分离的特征.
- 设计具有改变PAM特异性的FnCas9变体,并证明其在基因组编辑中的实用性.
主要方法:
- 使用X射线结晶学获得FnCas9指导RNA-DNA复合物的1.7 Å分辨率结构.
- 进行了FnCas9与其他已知的Cas9基因组之间的结构比较.
- 用定位突变生成来产生具有修改PAM识别的FnCas9变体.
- 将FnCas9核核蛋白复合体微注射到小鼠细胞中用于体内基因组编辑.
主要成果:
- 晶体结构显示FnCas9与其他Cas9正态相比保持和分离的特征.
- 发现FnCas9能够识别5'-NGG-3' PAM序列.
- 一种FnCas9的变异被设计为识别放松的5'-YG-3' PAM序列.
- 将工程FnCas9微注入小鼠囊中成功地修改了5'- YG-3' PAM的内源部位.
结论:
- 对FnCas9的结构洞察力提供了对CRISPR-Cas9系统多样性的更深入的理解.
- 设计FnCas9用于放松的PAM识别显著扩大了可向基因组位点的范围.
- 修改后的FnCas9系统有望在基因组编辑中得到更广泛的应用,包括哺乳动物系统.
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