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一种小型型V-N CRISPR-Cas12效应酶的机制和工程

Wenhan Fu¹, Jiacheng Ma¹, Zhipeng Wang¹

¹School of Physical Science and Technology & State Key Laboratory of Advanced Medical Materials and Devices, ShanghaiTech University, Shanghai, China.

Nature communications

|July 2, 2025

概括

研究人员阐明了微型CRISPR基因组编辑器Cas12n的结构和功能. 这项研究揭示了其独特的DNA向机制,并为设计新的Cas12n基因编辑工具铺平了道路.

科学领域:

分子生物学分子生物学
遗传学遗传学是一个
生物化学生物化学

背景情况:

类型V的CRISPR-Cas12系统,包括微型Cas12f1和Cas12n编辑器,由于它们的小尺寸,对于体内基因治疗至关重要.
Cas12f1核酶作为同位体,准富含T或C的PAMs,而Cas12n核酶可能是识别罕见的富含A的PAMs的单体.
由RNA引导的基因组向和Cas12n的分裂的精确分子机制在很大程度上仍未被描述.

研究的目的:

通过冷电子显微镜 (cryo-EM) 确定与sgRNA和点DNA结合的Rothia dentocariosa Cas12n (RdCas12n) 的结构.
阐明Cas12n的分子结构,PAM识别和核酸结合机制.
为V型CRISPR-Cas12系统的进化轨迹提供见解,并为基因组编辑应用程序设计Cas12n.

主要方法:

低温电子显微镜 (cryo-EM) 用于确定RdCas12n-sgRNA-DNA复合物的结构基础.
与其他Cas12核酶和TnpB进行结构比较,以推断进化关系.
在人类细胞中进行sgRNA修饰和功能测试,以设计RdCas12n作为基因组编辑器.

主要成果:

结合 sgRNA 和目标DNA 的 RdCas12n 的冷-EM 结构得到了解析,揭示了它的分子结构.
获得了Cas12n独特的A丰富的PAM识别和核酸结合机制的详细见解.
通过sgRNA修改,RdCas12n成功地被设计成人类细胞中的有效基因组编辑器.

结论:

这项研究提供了对Cas12n核酶的第一个结构见解,澄清了它们独特的准和结合机制.
进化比较凸显了Cas12n在V型CRISPR-Cas12系统多样化中的重要性.
经过工程设计的RdCas12n为开发基于Cas12n的新高效基因组编辑工具提供了一个有前途的分子基础.

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