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通过抗CRISPR蛋白抑制CRISPR-SpyCas9的结构基础

De Dong¹, Minghui Guo¹, Sihan Wang¹

¹HIT Center for Life Sciences, School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.

|April 28, 2017

概括

两种抗CRISPR蛋白通过模仿PAM和阻断DNA结合来抑制Cas9. 这种结构洞察力使得Cas9的发展成为可能.

科学领域:

分子生物学
结构生物学
遗传学

背景情况:

CRISPR-Cas9系统是针对外来DNA的细菌免疫机制.
Cas9酶使用导向RNA在特定部位分裂DNA,需要一个原体隔离基因 (PAM).
抗CRISPR蛋白AcrIIA2和AcrIIA4抑制Cas9,但它们的机制尚不清楚.

研究的目的:

阐明AcrIIA4抑制Streptococcus pyogenes Cas9 (SpyCas9) 的机制.
为开发可控制的Cas9系统提供结构基础.

主要方法:

用单导向RNA (sgRNA) 和AcrIIA4复合的SpyCas9的X射线晶体学
Cas9-sgRNA-AcrIIA4复合物的结构分析和比较.

主要成果:

AcrIIA4以SgRNA依赖的方式结合SpyCas9.
AcrIIA4通过模仿PAM并阻断DNA结合部位来抑制Cas9.
此外,AcrIIA4还保护了Cas9RuvC的活性部位,防止了DNA的裂变.
sgRNA结合诱导SpyCas9上的AcrIIA4结合部位的形成.

结论:

AcrIIA4通过一种双重机制抑制SpyCas9:阻断DNA识别并屏蔽活性部位.
这些发现为设计CRISPR-Cas9基因编辑工具的"关机"机制提供了结构基础.
了解这些相互作用对于精确的基因组编辑应用至关重要.

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