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为什么E1219V突变会扩大Streptococcus pyogenes的Cas9中的T-丰富的PAM识别?

Shreya Bhattacharya¹, Priyadarshi Satpati¹

¹Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India.

Journal of chemical information and modeling

|April 11, 2024

概括

计算机模拟揭示了Streptococcus pyogenes Cas9 (SpCas9) 突变如何改变DNA结合能量和PAM识别. 了解这些变化有助于设计改进的SpCas9基因组编辑工具.

科学领域:

分子生物学分子生物学
生物化学生物化学
结构生物学结构生物学

背景情况:

链球菌 pyogenes Cas9 (SpCas9) 是一种用于基因组编辑的关键RNA引导的DNA内核酶.
SpCas9识别了5'-NGG-3'原体空间器相邻动机 (PAM),以调解DNA裂变.
突变可以改变SpCas9的PAM识别,扩大其治疗应用的潜力,但潜在的能量和结构基础尚不清楚.

研究的目的:

调查SpCas9对PAM识别的能量及其与原子结构的关系.
阐明特定的SpCas9突变如何影响PAM结合亲和力和DNA裂变活性.
为合理设计SpCas9变体提供结构和热力学基础.

主要方法:

使用SpCas9:sgRNA:dsDNA的X射线结构作为模板.
运用计算机模拟来计算PAM结合亲和力的变化,以应对SpCas9突变.
分析了特定突变 (E1219V,R1335A) 以及它们对蛋白质-DNA相互作用和能量学的影响.

主要成果:

E1219V突变通过改变水的可访问性和促进与非正规PAMs的相互作用来削弱PAM严格性.
特定的氨酸残留物 (R1333,R1335) 对于严格的5'-NGG-3' PAM识别至关重要.
通过R1335A替代,通过破坏直接的PAM相互作用,取消了SpCas9编辑活动.
一种双重突变 (R1335A,E1219V) 通过在脱口袋中的静电相互作用增强了DNA结合亲和力.

结论:

这项研究确立了SpCas9变体的能量,原子结构和DNA裂变活性之间的直接联系.
热力学原理解释了在不同的SpCas9突变体中观察到的不同DNA裂变活动.
这些发现有助于合理设计基于SpCas9的增强基因组编辑技术.

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