Mechanistic studies of RNA-targeting CRISPR systems

摘要 细菌生命利用不同的免疫机制来保护自己免受捕食性噬菌体的侵害， 被认为是他们的十倍CRISPR系统特别涉及其组成Cas 核酸酶与可编程的指导RNA靶向入侵核酸，赋予宿主细胞 适应性免疫它们可以分为六大类型，VI型Cas13系统包含唯一的 已知的CRISPR核酸酶专门靶向RNA。CRISPR系统已被广泛应用， 基因工程技术，在过去的几年里，基于II型Cas9的平台 大大加快了基础研究和生物技术。就像Cas9用于DNA靶向一样，Cas13酶 可以被改造成一个模块化的高效平台，用于细胞中的RNA靶向， 操作工具箱。然而，许多Cas13酶受到可变和不可预测的活性的限制， 这一挑战限制了RNA干扰技术。更广泛地说， 基因组和转录组工程领域正在预测稳健的和可推广的切割效率， 在新开发的核酸酶效应物内，跨不同靶核酸和细胞类型的特异性。 最近，Hsu实验室报告发现了Cas13的一个亚型，Cas13d系统，这是显著的。 比其他Cas13亚型或短发夹RNA更小、更有效、更特异 选择性剪接的干扰和操纵。Lyumkis实验室最近利用了最先进的低温技术， 电子显微镜（cryo-EM）的进展，以解决与指导RNA结合的Cas13d的高分辨率结构 并靶向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Cas13d中观察到的分子/结构生物学之间的脱节。 哺乳动物细胞在转录组工程的努力。首要目标是阐明 CRISPR-Cas适应性免疫机制工程化改进的CRISPR相关酶基因 法规和其他生物技术应用。拟议的工作将系统地解决这些问题， 挑战使用跨学科的结构生物学，生物化学，蛋白质工程，生物信息学， Hsu和Lyumkis实验室合作的遗传方法。来自 拟议的工作将（1）提供对Cas13d的完整酶循环的机械见解，（2）阐明 Cas13d结构和功能的进化途径，（3）确定CRISPR的机制， 可以调节Cas13活性的相关因子，和（4）使下一个- 用于治疗和诊断应用的RNA靶向效应物的产生。重要的是，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.