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由盐和单导向RNA结合诱导的Streptococcus pyogenes Cas9的形状转变

Yufan He¹, Nikita Zalenski¹, Anthony A Stephenson²

¹Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida, USA.

The Journal of biological chemistry

|December 24, 2024

概括

这项研究揭示了Streptococcus pyogenes Cas9蛋白质存在于不同的构造,受盐度和指导RNA存在的影响. 这表明Cas9使用了对指导RNA结合的 conformational 采样.

科学领域:

分子生物学分子生物学
生物化学生物化学
遗传学遗传学是一个

背景情况:

Streptococcus pyogenes Cas9 (SpCas9) 是基因组编辑的一个关键酶.
对于有效的DNA向和裂变,SpCas9需要特定的结构变化.
了解这些动态对于优化基因编辑技术至关重要.

研究的目的:

为了研究SpCas9.9的结构动态.
为了确定化 (KCl) 度对SpCas9形状的影响.
探索KCl在SpCas9-sgRNA相互作用中的作用.

主要方法:

采用了单分子福斯特共振能量转移 (smFRET) 技术.
在不同的KCl度下分析了SpCas9的形态状态.
实验包括使用和不使用单导向RNA (sgRNA) 的实验.

主要成果:

在没有sgRNA的情况下,ApoCas9采用了两种构造:一种自抑制的开放状态 (Cas9^apo) 和一种与sgRNA结合的类似状态 (Cas9^X).
增加KCl度有利于Cas9^X形状,同时减少Cas9^apo群体.
相反,在sgRNA的存在下,KCl对Cas9^X和Cas9^apo群体产生了相反的影响.

结论:

KCl度作为SpCas9形态动态的探测器.
研究结果表明,SpCas9可能利用sgRNA结合的 conformational 采样机制.
这补充了先前建立的SpCas9-sgRNA相互作用的诱导适合模型.

关键词:

克里斯普尔卡斯9 构造动力学构造动力学基因编辑酶的基因编辑酶.诱导适应机制的诱导适应机制单分子FRETET是一种单分子FRET.

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