National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894, United States.
McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
Biochemistry. 2023 Dec 19;62(24):3465-3487. doi: 10.1021/acs.biochem.3c00159. Epub 2023 May 16.
CRISPR systems mediate adaptive immunity in bacteria and archaea through diverse effector mechanisms and have been repurposed for versatile applications in therapeutics and diagnostics thanks to their facile reprogramming with RNA guides. RNA-guided CRISPR-Cas targeting and interference are mediated by effectors that are either components of multisubunit complexes in class 1 systems or multidomain single-effector proteins in class 2. The compact class 2 CRISPR systems have been broadly adopted for multiple applications, especially genome editing, leading to a transformation of the molecular biology and biotechnology toolkit. The diversity of class 2 effector enzymes, initially limited to the Cas9 nuclease, was substantially expanded via computational genome and metagenome mining to include numerous variants of Cas12 and Cas13, providing substrates for the development of versatile, orthogonal molecular tools. Characterization of these diverse CRISPR effectors uncovered many new features, including distinct protospacer adjacent motifs (PAMs) that expand the targeting space, improved editing specificity, RNA rather than DNA targeting, smaller crRNAs, staggered and blunt end cuts, miniature enzymes, promiscuous RNA and DNA cleavage, etc. These unique properties enabled multiple applications, such as harnessing the promiscuous RNase activity of the type VI effector, Cas13, for supersensitive nucleic acid detection. class 1 CRISPR systems have been adopted for genome editing, as well, despite the challenge of expressing and delivering the multiprotein class 1 effectors. The rich diversity of CRISPR enzymes led to rapid maturation of the genome editing toolbox, with capabilities such as gene knockout, base editing, prime editing, gene insertion, DNA imaging, epigenetic modulation, transcriptional modulation, and RNA editing. Combined with rational design and engineering of the effector proteins and associated RNAs, the natural diversity of CRISPR and related bacterial RNA-guided systems provides a vast resource for expanding the repertoire of tools for molecular biology and biotechnology.
CRISPR 系统通过多种效应机制介导细菌和古菌的适应性免疫，并且由于其易于用 RNA 向导进行重新编程，因此已被重新用于治疗和诊断中的多种应用。RNA 引导的 CRISPR-Cas 靶向和干扰由效应子介导，这些效应子要么是 1 类系统中的多亚基复合物的组成部分，要么是 2 类中的多结构域单效蛋白。紧凑的 2 类 CRISPR 系统已被广泛用于多种应用，特别是基因组编辑，从而改变了分子生物学和生物技术工具包。最初仅限于 Cas9 核酸酶的 2 类效应酶的多样性通过计算基因组和宏基因组挖掘得到了极大扩展，包括 Cas12 和 Cas13 的许多变体，为多功能、正交分子工具的开发提供了底物。对这些不同的 CRISPR 效应子的表征揭示了许多新特性，包括扩展靶向空间的独特的前导序列相邻基序 (PAM)、提高的编辑特异性、RNA 而不是 DNA 靶向、更小的 crRNA、交错和钝端切割、微型酶、混杂的 RNA 和 DNA 切割等。这些独特的特性实现了多种应用，例如利用 VI 型效应子 Cas13 的混杂 RNase 活性进行超灵敏核酸检测。尽管表达和递送多蛋白 1 类效应子具有挑战性，但 1 类 CRISPR 系统也已被用于基因组编辑。CRISPR 酶的丰富多样性导致基因组编辑工具包的快速成熟，具有基因敲除、碱基编辑、prime 编辑、基因插入、DNA 成像、表观遗传调节、转录调节和 RNA 编辑等功能。与效应蛋白和相关 RNA 的合理设计和工程相结合，CRISPR 和相关细菌 RNA 引导系统的自然多样性为扩展分子生物学和生物技术的工具库提供了巨大的资源。
