RNA targeting tools with novel specific RNA-guided RNA-targeting CRISPR effectors

项目摘要 尽管基因组工程取得了非凡的进步，但精确和高效的转录组工程工具 都是缺乏的。虽然我们和其他人已经表征了针对CRISPR系统的新型可编程RNA，例如 作为Cas13，以及从这些系统开发的工具，这些工具在蜂窝系统中的使用由于 一种称为附属活动的非混杂切割活动，Cas13的主要应用是 快速、灵敏的核酸检测，利用侧枝活动产生报告信号。而Cas13 已被证明在某些类型的细胞中具有特定的RNA切割活性，其他类型的细胞具有显著的 细胞RNA的侧枝裂解，导致细胞模型的毒性。一种理想的哺乳动物RNA靶向工具 应用将缺乏附带活性，并且只切割目标底物。拟议的工作将解决 通过结合计算发现、生物化学特性和酶工程来满足这些需求 发现新的针对CRISPR核酸酶的RNA，使这些酶适应哺乳动物的使用，并开发出特定的 转录组工程和全转录组筛选的RNA靶向工具。这一发现和 这些新的CRISPR蛋白的表征将建立在我们对CRISPR酶工作的深厚历史的基础上， 以及利用新的高通量方法开采生物多样性。我们将寻找家庭 在CRISPR阵列附近富含核糖核酸酶结构域，并对这些酶进行表征。初步 一个含有核糖核酸酶家族，含有类Cas7的RAMP核糖核酸酶结构域，这里称为 Cas7-11，显示了使用短引导RNA对特定靶点的RNA切割，而没有观察到侧枝活性。 该Cas7-11效应器属于III-E型系统，是第一个具有特征化的单蛋白效应器 1个系统。我们表征了Cas7-11的作用机制，展示了使其不具有催化活性的残基 RNA结合应用，以及在哺乳动物细胞中进行RNA敲除和编辑的工程师Cas7-11。使用 具体的Cas7-11工具，我们建议开发一种能够击倒RNA的单一技术，即RNA 编辑，或基于crRNA的RNA拼接，允许在单个 全基因组RNA靶向屏幕，并允许有效地询问细胞电路。多面性 由这些发现和工程工作产生的技术将克服现有技术的限制 转录组工程方法，并作为更广泛的生物医学研究的宝贵资源。 此外，这种基因探索和工程框架将作为发现多样性的模型。 细菌基因，评估一系列检测的生化活性，并将这些发现转化为高 影响生物技术。开发的技术将加快生物医学研究的步伐，并使 更多地探索基本的生物学过程和疾病机制。

Project Summary Despite extraordinary advances in genome engineering, tools for precise and efficient transcriptome engineering are lacking. While we and others have characterized novel programmable RNA targeting CRISPR systems, such as Cas13, and developed tools from these systems, use of these tools have been limited in cellular systems due to a non-promiscuous cleavage activity known as collateral activity, and the main application for Cas13 has been rapid and sensitive nucleic acid testing using the collateral activity for reporter signal generation. While Cas13 has been shown to have specific RNA cleavage activity in some cell types, other cell types have had significant collateral cleavage of cellular RNAs, leading to toxicity in cell models. An ideal RNA targeting tool for mammalian applications would lack collateral activity and only cleave the targeted substrate. The proposed work will address these needs by combining computational discovery, biochemical characterization, and enzyme engineering to find new RNA targeting CRISPR nucleases, adapt these enzymes for mammalian use, and develop specific RNA targeting tools for transcriptome engineering and transcriptome-wide screening. The discovery and characterization of these new CRISPR proteins will both build upon our deep history of CRISPR enzyme work, as well as draw from new, high-throughput approaches to mine biological diversity. We will search for families with RNase domains enriched near CRISPR arrays and characterize these enzymes. Preliminary characterization of one RNase containing family, containing Cas7-like RAMP RNase domains, here termed Cas7-11, shows RNA cleavage of specific targets using short guide RNAs without observable collateral activity. This Cas7-11 effector belongs to type III-E systems and is the first characterized single-protein effector in Class 1 systems. We characterize the mechanism of Cas7-11, show the residues that make it catalytically inactive for RNA binding applications, and engineer Cas7-11 for RNA knockdown and editing in mammalian cells. Using the specific Cas7-11 tool, we propose developing a single technology that is capable of RNA knockdown, RNA editing, or RNA splicing based on the crRNA, allowing multiple RNA perturbations to be accomplished in a single genome wide RNA targeting screen and allowing for cell circuits to be efficiently interrogated. The multiple technologies resulting from these discoveries and engineering efforts will overcome the limitations of existing transcriptome engineering approaches and serve as a valuable resource for broader biomedical research. Moreover, this gene exploration and engineering framework will serve as a model for discovering diverse bacterial genes, evaluating biochemical activity across a range of assays, and converting these findings into high impact biotechnologies. The developed technologies will accelerate the pace of biomedical research and enable greater exploration of basic biological processes and disease mechanisms.