*Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, U.S.A.
Biochem Soc Trans. 2013 Dec;41(6):1416-21. doi: 10.1042/BST20130056.
Using the hyperthermophile Pyrococcus furiosus, we have delineated several key steps in CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) invader defence pathways. P. furiosus has seven transcriptionally active CRISPR loci that together encode a total of 200 crRNAs (CRISPR RNAs). The 27 Cas proteins in this organism represent three distinct pathways and are primarily encoded in two large gene clusters. The Cas6 protein dices CRISPR locus transcripts to generate individual invader-targeting crRNAs. The mature crRNAs include a signature sequence element (the 5' tag) derived from the CRISPR locus repeat sequence that is important for function. crRNAs are tailored into distinct species and integrated into three distinct crRNA-Cas protein complexes that are all candidate effector complexes. The complex formed by the Cmr [Cas module RAMP (repeat-associated mysterious proteins)] (subtype III-B) proteins cleaves complementary target RNAs and can be programmed to cleave novel target RNAs in a prokaryotic RNAi-like manner. Evidence suggests that the other two CRISPR-Cas systems in P. furiosus, Csa (Cas subtype Apern) (subtype I-A) and Cst (Cas subtype Tneap) (subtype I-B), target invaders at the DNA level. Studies of the CRISPR-Cas systems from P. furiosus are yielding fundamental knowledge of mechanisms of crRNA biogenesis and silencing for three of the diverse CRISPR-Cas pathways, and reveal that organisms such as P. furiosus possess an arsenal of multiple RNA-guided mechanisms to resist diverse invaders. Our knowledge of the fascinating CRISPR-Cas pathways is leading in turn to our ability to co-opt these systems for exciting new biomedical and biotechnological applications.
我们利用嗜热古菌 Pyrococcus furiosus，阐明了 CRISPR（成簇规律间隔短回文重复）-Cas（CRISPR 相关）入侵防御途径中的几个关键步骤。P. furiosus 有七个转录活性的 CRISPR 基因座，总共编码 200 个 crRNA（CRISPR RNA）。该生物体中的 27 种 Cas 蛋白代表三种不同的途径，主要编码在两个大的基因簇中。Cas6 蛋白将 CRISPR 基因座转录本切割成单独的靶向入侵体的 crRNA。成熟的 crRNA 包含一个来自 CRISPR 基因座重复序列的特征序列元件（5'标签），这对于功能很重要。crRNA 被剪裁成不同的物种，并整合到三个不同的 crRNA-Cas 蛋白复合物中，这些复合物都是候选效应复合物。由 Cmr（Cas 模块 RAMP（重复相关神秘蛋白））（亚型 III-B）蛋白组成的复合物切割互补的靶 RNA，并可以通过类似于原核 RNAi 的方式编程切割新的靶 RNA。有证据表明，P. furiosus 中的另外两种 CRISPR-Cas 系统，Csa（Cas 亚型 Apern）（亚型 I-A）和 Cst（Cas 亚型 Tneap）（亚型 I-B），在 DNA 水平上靶向入侵体。对 P. furiosus 的 CRISPR-Cas 系统的研究为三种不同的 CRISPR-Cas 途径的 crRNA 生物发生和沉默机制提供了基础知识，并揭示了像 P. furiosus 这样的生物体拥有多种 RNA 指导的机制来抵抗不同的入侵体。我们对迷人的 CRISPR-Cas 途径的了解反过来又使我们能够利用这些系统进行令人兴奋的新的生物医学和生物技术应用。
