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结合计算和实验方法,以扩大DNA准范围来探索SaCas9的分子机制

Binquan Luan¹, Guangxue Xu², Mei Feng³

¹Computational Biological Center , IBM Thomas J. Watson Research , Yorktown Heights , New York 10598 , United States.

Journal of the American Chemical Society

|March 30, 2019

概括

研究人员通过研究 Staphylococcus aureus Cas9 (SaCas9) 酶来探索CRISPR-Cas9技术. 他们揭示了SaCas9变体的分子机制,从而设计出具有更强活力和更广泛准能力的新突变体.

科学领域:

生物技术
分子生物学
基因组学

背景情况:

尽管CRISPR技术正在迅速发展,
工程 CRISPR 工具通常缺乏对其改进特性的机械解释.
黄金葡萄球菌Cas9 (SaCas9) 适用于体内应用,但需要长的原体隔离基因 (PAM).

研究的目的:

研究SaCas9 PAM识别的分子动力学.
阐明SaCas9中KKH突变的机制.
设计和验证具有增强性能的新型SaCas9变种.

主要方法:

基于结构的分子动力学模拟.
自由能量扰动分析.
针对 SaCas9 变种进行实验验证.

主要成果:

在SaCas9中揭示了PAM识别的动态机制.
阐明了KKH突变的分子基础.
设计和验证的SaCas9-NR和SaCas9-RL突变体具有扩大向范围和增强哺乳动物细胞中的活性.

结论:

这项研究提供了对SaCas9 PAM识别的动态理解.
开发的工作流程可以合理设计具有新特性的Cas9变体.
新的SaCas9变种为基因编辑应用提供了改进的工具.

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