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Updated: Jun 13, 2025

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

通过抑制复制,CRISPR-Cas9的目标链切割提供了菌体的耐药性

Giang T Nguyen¹, Michael A Schelling¹, Dipali G Sashital¹

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

bioRxiv : the preprint server for biology

|September 16, 2024

概括

克里斯普尔Cas9尼克酶可以通过切断目标DNA来提供细菌免疫力,即使没有完全分裂它. 这种DNA切割抑制了菌体的复制,特别是在低离子度下.

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.
遗传学是一个遗传学.

背景情况:

卡斯核内核酶 (例如,Cas9,Cas12a) 是对菌体的关键细菌免疫作用因子.
这些酶需要双价金属离子,如Mg2+,用于DNA结合和裂变.
凯斯酶通常会诱导双链断裂 (DSB),但在某些条件下可以发生断.

研究的目的:

调查Cas内核酶对DNA的破坏是否足以产生菌体免疫力.
为了确定目标链与非目标链在细菌防御中的作用.
了解离子度如何影响Cas9裂变活性和细菌防御.

主要方法:

使用一个活性核酶域的Cas9尼克酶.
测试了广泛的导向RNA,以测试目标DNA裂变的效率.
通过不同的Cas9-介导的DNA修饰结果赋予的评估菌体保护.
研究了不同离子度对Cas9活性的影响.

主要成果:

通过Cas9的杀目标链足以赋予菌体免疫力.
切断非目标链除了酶结合之外,没有提供额外的保护.
低度的Mg2+显著损害了RuvC域的非目标链裂变.
目标链切割抑制了菌体的复制,降低了病毒逃生潜力.

结论:

在CRISPR-Cas免疫力方面,Cas9介导的DNA切割可能是足够的.
细胞Mg2+度极大地影响了Cas9诱导DSB与nicks的能力.
在自然的CRISPR介导免疫中,Cas9可能主要作为一个尼克酶,特别是在低Mg2+的环境中.

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