Mechanistic studies of RNA-targeting CRISPR systems

摘要 细菌生命使用不同的免疫机制来保护自己免受捕食性噬菌体的攻击，这些机制包括 被认为是他们的十倍。CRISPR系统尤其涉及其组成的CA 核酸酶和可编程的引导RNA以入侵的核酸为靶标，赋予宿主细胞 适应性免疫。它们可以分为六大类型，而类型VI Cas13系统仅包含 已知的CRISPR核酸酶，专门针对RNA。CRISPR系统已被广泛采用为 基因工程技术，在过去的几年里，基于类型II Cas9的平台已经 显著加快基础研究和生物技术建设。很像用于DNA靶向的Cas9，Cas13酶 可以被改造成一个模块化的高效平台，用于在细胞中进行RNA靶向，极大地促进了RNA 操纵工具箱。然而，许多Cas13酶受到可变和不可预测的活性的限制，a 这一挑战限制了RNA干扰技术。更广泛地说，一个核心问题是 基因组和转录组工程领域正在预测稳健和普遍的切割效率和 在新开发的核酸酶效应器中，跨不同目标核酸和细胞类型的特异性。 最近，徐实验室报告发现了Cas13的一个亚型，Cas13d系统，这对 比其他Cas13亚型或短发夹状RNA更小、更有效、更特异 交替剪接的干涉和操纵。Lyumkis实验室最近利用最先进的冷冻机- 电子显微镜(Cryo-EM)在解决Cas13d与引导RNA结合的高分辨结构方面的进展 和靶向RNA。然而，我们对Cas13d分子结构和功能的理解存在差距。 定义Cas13d活性的分子/结构生物学与在 哺乳动物细胞在转录组工程方面的努力。最重要的目标是阐明 CRISPR-Cas获得性免疫基因工程改良CRISPR相关酶的机制 监管和其他生物技术应用。拟议的工作将系统地解决这些问题 使用跨学科的结构生物学、生化、蛋白质工程、生物信息学和 徐氏实验室和Lyumkis实验室合作的遗传方法。综合结果来自 拟议的工作将(1)提供对Cas13d完整的酶循环的机械性见解，(2)揭示 关于参与Cas13d结构和功能的进化途径，(3)确定了CRISPR的机制-- 相关因素，可以调节Cas13的活性，以及(4)使结构指导的工程下一步- 产生用于治疗和诊断应用的RNA靶向效应器。重要的是，这些原则和 这里阐述的方法将为未来各种工具的设计提供蓝图 CRISPR-CAS13是一个全面的基因组工程工具箱。

Abstract Bacterial life employs diverse immune mechanisms to protect themselves against predatory phage, which are thought to outnumber them by ten to one. CRISPR systems in particular engage their constituent Cas nucleases with programmable guide RNAs to target invading nucleic acids, endowing the host cell with adaptive immunity. They can be divided into six broad types, and the Type VI Cas13 systems contain the only known CRISPR nucleases that exclusively target RNA. CRISPR systems have been broadly adapted as genetic engineering technologies, and over the last few years, platforms based on Type II Cas9 have significantly accelerated basic research and biotechnology. Much like Cas9 for DNA targeting, Cas13 enzymes can be adapted into a modular and efficient platform for RNA targeting in cells, greatly advancing the RNA manipulation toolbox. However, many Cas13 enzymes are limited by variable and unpredictable activity, a challenge that has limited RNA interference technologies. More broadly speaking, a central problem in the genome and transcriptome engineering field is predicting robust and generalizable cleavage efficiency and specificity across different target nucleic acids and cell types within newly developed nuclease effectors. Recently, the Hsu lab reported the discovery of a subtype of Cas13, the Cas13d system, which is significantly smaller, more efficient, and more specific than other Cas13 subtypes or short hairpin RNAs for RNA interference and manipulation of alternative splicing. The Lyumkis lab recently leveraged state-of-the-art cryo- electron microscopy (cryo-EM) advances to solve high-resolution structures of Cas13d bound to guide RNA and target RNA. However, there are gaps in our understanding of Cas13d molecular structure and function and disconnects between the molecular/structural biology defining Cas13d activity and what is observed in mammalian cells in transcriptome engineering efforts. The overarching goal is to elucidate the diverse mechanisms of CRISPR-Cas adaptive immunity to engineer improved CRISPR-associated enzymes for gene regulation and other biotechnological applications. The proposed work will systematically address these challenges using interdisciplinary structural biology, biochemical, protein engineering, bioinformatic, and genetic approaches in collaboration between the Hsu and Lyumkis labs. The combined results from the proposed work will (1) provide mechanistic insight into the complete enzymatic cycle of Cas13d, (2) shed light on the evolutionary pathways involved in Cas13d structure and function, (3) define the mechanism of CRISPR- associated factors that can modulate Cas13 activity, and (4) enable the structure-guided engineering of next- generation RNA-targeting effectors for therapeutic and diagnostic applications. Importantly, the principles and approaches elucidated here will provide a blueprint for the design of diverse forthcoming tools beyond CRISPR-Cas13 for a comprehensive genome engineering toolbox.