最小型 VI CRISPR-Cas13bt3 的结构与工程
Structure and engineering of the minimal type VI CRISPR-Cas13bt3.
机构信息
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
出版信息
Mol Cell. 2022 Sep 1;82(17):3178-3192.e5. doi: 10.1016/j.molcel.2022.08.001. Epub 2022 Aug 25.
Type VI CRISPR-Cas13 effector enzymes catalyze RNA-guided RNA cleavage and have been harnessed for various technologies, such as RNA detection, targeting, and editing. Recent studies identified Cas13bt3 (also known as Cas13X.1) as a miniature Cas13 enzyme, which can be used for knockdown and editing of target transcripts in mammalian cells. However, the action mechanism of the compact Cas13bt3 remains unknown. Here, we report the structures of the Cas13bt3-guide RNA complex and the Cas13bt3-guide RNA-target RNA complex. The structures revealed how Cas13bt3 recognizes the guide RNA and its target RNA and provided insights into the activation mechanism of Cas13bt3, which is distinct from those of the other Cas13a/d enzymes. Furthermore, we rationally engineered enhanced Cas13bt3 variants and ultracompact RNA base editors. Overall, this study improves our mechanistic understanding of the CRISPR-Cas13 enzymes and paves the way for the development of efficient Cas13-mediated transcriptome modulation technologies.
VI 型 CRISPR-Cas13 效应酶催化 RNA 引导的 RNA 切割，已被广泛应用于 RNA 检测、靶向和编辑等各种技术中。最近的研究鉴定出 Cas13bt3（也称为 Cas13X.1）作为一种微型 Cas13 酶，可用于哺乳动物细胞中靶转录本的敲低和编辑。然而，紧凑型 Cas13bt3 的作用机制尚不清楚。在这里，我们报告了 Cas13bt3 指导 RNA 复合物和 Cas13bt3 指导 RNA-靶 RNA 复合物的结构。这些结构揭示了 Cas13bt3 如何识别指导 RNA 及其靶 RNA，并提供了对 Cas13bt3 激活机制的深入了解，该机制与其他 Cas13a/d 酶的机制不同。此外，我们还进行了合理的工程设计，增强了 Cas13bt3 变体和超紧凑的 RNA 碱基编辑器。总的来说，这项研究提高了我们对 CRISPR-Cas13 酶的机制理解，并为开发高效的 Cas13 介导的转录组调节技术铺平了道路。
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