Structure of the miniature type V-F CRISPR-Cas effector enzyme
Structure of the miniature type V-F CRISPR-Cas effector enzyme
复制标题
DOI：
10.1016/j.molcel.2020.11.035
复制
发表时间：
2021-02-04
期刊：
影响因子：
16
通讯作者：
Nureki, Osamu
中科院分区：
文献类型：
--
作者：
Takeda, Satoru N.;Nakagawa, Ryoya;Nureki, Osamu
来源于CRISPR-Cas适应性免疫系统的RNA引导的DNA内切酶被广泛用作强大的基因组工程工具。在各种CRISPR-Cas核酸酶中，V-F Cas12f(也称为Cas14)蛋白非常紧凑，与引导RNA结合，切割单链和双链DNA靶标。在这里，我们报告了Cas12f1(也称为Cas14a)的冷冻电子显微镜结构，它与引导RNA及其靶DNA形成了复合体。出乎意料的是，结构显示两个Cas12f1分子与单个引导RNA组装在一起，识别双链DNA靶标。每个Cas12f1原核酸体采用不同的构象，在核酸识别和DNA切割中发挥不同的作用，从而解释了微型Cas12f1酶是如何作为“不对称同源二聚体”实现RNA引导的DNA切割的。我们的发现增强了对不同CRISPR-CAS核酸酶的机械理解，并为开发对治疗性基因组编辑至关重要的紧凑基因组工程工具提供了一个框架。
RNA-guided DNA endonucleases derived from CRISPR-Cas adaptive immune systems are widely used as powerful genome-engineering tools. Among the diverse CRISPR-Cas nucleases, the type V-F Cas12f (also known as Cas14) proteins are exceptionally compact and associate with a guide RNA to cleave single and double-stranded DNA targets. Here, we report the cryo-electron microscopy structure of Cas12f1 (also known as Cas14a) in complex with a guide RNA and its target DNA. Unexpectedly, the structure revealed that two Cas12f1 molecules assemble with the single guide RNA to recognize the double-stranded DNA target. Each Cas12f1 protomer adopts a different conformation and plays distinct roles in nucleic acid recognition and DNA cleavage, thereby explaining how the miniature Cas12f1 enzyme achieves RNA-guided DNA cleavage as an "asymmetric homodimer." Our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and provide a framework for the development of compact genome-engineering tools critical for therapeutic genome editing.