Omura Satoshi N, Nureki Osamu
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Methods Enzymol. 2025;712:23-39. doi: 10.1016/bs.mie.2025.01.052. Epub 2025 Feb 19.
Cas9 and Cas12 are RNA-guided DNA endonucleases derived from prokaryotic CRISPR-Cas adaptive immune systems that have been repurposed as versatile genome-engineering tools. Computational mining of genomes and metagenomes has expanded the diversity of Cas9 and Cas12 enzymes that can be used to develop versatile, orthogonal molecular toolboxes. Structural information is pivotal to uncovering the precise molecular mechanisms of newly discovered Cas enzymes and providing a foundation for their application in genome editing. In this chapter, we describe detailed protocols for the preparation of Cas9 and Cas12 enzymes for cryo-electron microscopy. These methods will enable fast and robust structural determination of newly discovered Cas9 and Cas12 enzymes, which will enhance the understanding of diverse CRISPR-Cas effectors and provide a molecular framework for expanding CRISPR-based genome-editing technologies.
Cas9和Cas12是源自原核CRISPR-Cas适应性免疫系统的RNA引导的DNA核酸内切酶，已被重新用作通用的基因组工程工具。对基因组和宏基因组的计算挖掘扩展了Cas9和Cas12酶的多样性，这些酶可用于开发通用的、正交的分子工具箱。结构信息对于揭示新发现的Cas酶的精确分子机制以及为其在基因组编辑中的应用提供基础至关重要。在本章中，我们描述了用于冷冻电子显微镜的Cas9和Cas12酶制备的详细方案。这些方法将能够快速、稳健地确定新发现的Cas9和Cas12酶的结构，这将增进对各种CRISPR-Cas效应物的理解，并为扩展基于CRISPR的基因组编辑技术提供分子框架。
