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用5'截断的sgRNA进行Cas9DNA查询的结构基础

Kaitlyn A Kiernan¹, Jieun Kwon¹, Bradley J Merrill¹

¹Department of Biochemistry and Molecular Genetics, University of Illinois Chicago, 900 S Ashland Ave, Chicago, IL 60607, USA.

Nucleic acids research

|December 10, 2024

概括

较短的单导向RNAs (sgRNAs) 通过改变Cas9结构来增强CRISPR-Cas9的特异性,防止核酶域的激活. 这种结构洞察力解释了切断的sgRNA如何提高基因组应用中的目标准确性.

科学领域:

分子生物学分子生物学
生物化学生化学
结构生物学结构生物学

背景情况:

CRISPR-Cas9基因编辑效率和准确性受到目标变异性的限制.
5'截断的单导向RNAs (sgRNAs) 在保持特异性的同时,有望减少非目标效应.
截断的sgRNA增强特异性并影响Cas9激活的机制尚不清楚.

研究的目的:

研究CRISPR-Cas9活动与5'截断的sgRNAs的结构和生化基础.
了解 sgRNA 截断如何影响 Cas9 的结合,激活和特异性.
阐明非目标链 (NTS) 在Cas9函数中的作用.

主要方法:

生物化学分析测量Cas9活性和结合.
低温电子显微镜 (cryo-EM) 用于确定Cas9-sgRNA复杂结构.
对Cas9-sgRNA相互作用和核酶域定位的分析.

主要成果:

冷-EM结构显示,14-nt sgRNA 由于缩短的 NTS 路径,导致 HNH L1 链接器的硬质阻碍.
这种固态碰撞阻止了Cas9核酶域的正确定位,抑制了裂变.
切割抑制可以通过减轻双重菌株或使用超卷DNA来克服,但等离子体切割速度比全长sgRNA慢1000倍.

结论:

这项研究为5'截断的sgRNAs减少的Cas9活性提供了结构性解释.
PAM-遥远NTS的可用性对于促进Cas9激活和高效的DNA裂变至关重要.
这些发现强调了sgRNA长度和NTS相互作用对于精确的CRISPR-Cas9基因组编辑的重要性.

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