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快速的CRISPR-Cas9目标链切割可以通过减少DNA丰度来提供菌体抵抗力

Giang T Nguyen¹, Akshara Raju¹, Michael A Schelling¹

¹Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, United States.

Nucleic acids research

|September 23, 2025

概括

细菌Cas9 (CRISPR相关蛋白9) 免疫力依赖于其HNH域的目标链切断,而不是双链DNA断裂. 这种切割可以防止菌体DNA的积累和病毒逃逸,即使含量有限.

科学领域:

分子生物学分子生物学
细菌学细菌学是一门学科.
生物化学生化学

背景情况:

Cas9是一种细菌免疫内核酶,对抗菌体的防御至关重要.
Cas9利用双价金属离子,如 (Mg2+),通过其HNH和RuvC域进行DNA裂变催化.
生理学Mg2+度可以影响Cas9的裂变活动,可能有利于在双链断裂 (DSB) 上进行切割.

研究的目的:

为了调查Cas9 HNH域单独对菌体产生细菌免疫力是否足够.
在不同的Mg2+条件下确定RuvC域在Cas9介导的菌体防御中的作用.
了解生物分子相互作用如何影响Cas9的分裂机制及其在免疫中的有效性.

主要方法:

利用具有HNH或RuvC域功能的催化活性Cas9尼克酶来评估个别域的贡献.
在生理学上相关的Mg2+度下测试了这些Cas9尼克酶的菌体保护功效.
分析了竞争生物分子对Cas9的RuvC介导裂变和DSB形成的影响.

主要成果:

HNH域的Cas9杀活性,但不是RuvC域,赋予了足够的菌体免疫力.
目标链切割有效抑制了菌体DNA的积累,并减少了病毒逃逸.
由RuvC介导的DSB形成受到竞争生物分子的显著阻碍,这些生物分子结合自由Mg2+.

结论:

对于CRISPR介导的对菌体的细菌免疫力来说,HNH域的目标链切割足够.
在生理条件下,HNH裂变似乎比RuvC裂变更快,从而启动免疫力.
在复杂的生物环境中,Cas9的免疫机制可能会优先考虑在复杂的生物环境中对DSB的形成进行切割.

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