RNA-guided binding and cleavage of nucleic acids by CRISPR–Cas systems is a defining feature of bacterial and archaeal adaptive immunity against viruses and plasmids. As a result of their programmable ability to cut specific DNA and RNA sequences, Cas9 and related single-subunit effector proteins from CRISPR–Cas systems have been widely adopted for research and therapeutic genome engineering applications. In this Review, we discuss the chemistry of macromolecules involved in the multistep interference pathway used by CRISPR–Cas systems that mediate accurate nucleic acid target recognition and cutting. Although this Review mainly focuses on DNA interference by Cas9, we briefly explore nucleic acid targeting by the single-effector proteins Cas12 and Cas13 to emphasize the conserved themes of precision DNA and RNA cleavage within class 2 CRISPR–Cas systems. We further highlight the unique mechanisms of surveillance complex formation, substrate recognition and target cleavage in molecular detail across diverse single-subunit CRISPR–Cas interference proteins.

CRISPR–Cas系统的RNA引导结合和核酸切割是细菌和古细菌对病毒和质粒的适应性免疫的主要特征。由于其可编程的剪切特定DNA和RNA序列的能力，CRISPR-Cas系统的Cas9和相关单亚基效应蛋白已被广泛用于研究和基因组工程应用。在这篇综述中，我们讨论了CRISPR-Cas系统所使用的多步干扰途径中涉及的大分子化学，其介导了准确的核酸靶标识别和切割。尽管本评论主要关注Cas9对DNA的干扰，我们简要地探讨了单个效应蛋白Cas12和Cas13的核酸靶向，以强调2类CRISPR-Cas系统内精确的DNA和RNA裂解的保守主题。我们进一步在各种单亚基CRISPR–Cas干扰蛋白的分子细节中重点介绍了监视复合物形成，底物识别和靶标裂解的独特机制。