Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA; Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA.
J Mol Biol. 2019 Jan 4;431(1):66-87. doi: 10.1016/j.jmb.2018.06.029. Epub 2018 Jun 22.
Prokaryotic adaptive immune systems use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated (Cas) proteins for RNA-guided cleavage of foreign genetic elements. The focus of this review, Type VI CRISPR-Cas systems, contain a single protein, Cas13 (formerly C2c2) that when assembled with a CRISPR RNA (crRNA) forms a crRNA-guided RNA-targeting effector complex. Type VI CRISPR-Cas systems can be divided into four subtypes (A-D) based on Cas13 phylogeny. All Cas13 proteins studied to date possess two enzymatically distinct ribonuclease activities that are required for optimal interference. One RNase is responsible for pre-crRNA processing to form mature Type VI interference complexes, while the other RNase activity provided by the two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, is required for degradation of target-RNA during viral interference. In this review, I will compare and contrast what is known about the molecular architecture and behavior of Type VI (A-D) CRISPR-Cas13 interference complexes, how this allows them to carry out their RNA-targeting function, how Type VI accessory proteins are able to modulate Cas13 activity, and how together all of these features have led to the rapid development of a range of RNA-targeting applications. Throughout I will also discuss some of the outstanding questions regarding Cas13's molecular behavior, and its role in bacterial adaptive immunity and RNA-targeting applications.
原核生物适应性免疫系统利用成簇规律间隔短回文重复序列 (CRISPRs) 和 CRISPR 相关 (Cas) 蛋白，实现对外源遗传元件的 RNA 引导切割。本综述的重点是 VI 型 CRISPR-Cas 系统，它包含一个单一的蛋白质 Cas13（以前称为 C2c2），当与 CRISPR RNA (crRNA) 组装时，形成一个 crRNA 引导的 RNA 靶向效应复合物。根据 Cas13 的系统发育，VI 型 CRISPR-Cas 系统可分为四个亚型 (A-D)。迄今为止研究过的所有 Cas13 蛋白都具有两种不同的酶促核糖核酸酶活性，这是最佳干扰所必需的。一种 RNase 负责 pre-crRNA 加工以形成成熟的 VI 型干扰复合物，而另一种由两个高等真核生物和原核生物核苷酸结合 (HEPN) 结构域提供的 RNase 活性，是在病毒干扰过程中降解靶 RNA 所必需的。在这篇综述中，我将比较和对比 VI 型 (A-D) CRISPR-Cas13 干扰复合物的分子结构和行为，以及它们如何执行其 RNA 靶向功能，VI 型辅助蛋白如何调节 Cas13 的活性，以及所有这些特征如何共同导致一系列 RNA 靶向应用的快速发展。在整个过程中，我还将讨论一些关于 Cas13 分子行为及其在细菌适应性免疫和 RNA 靶向应用中的作用的悬而未决的问题。
