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

具有新型特异性 RNA 引导 RNA 靶向 CRISPR 效应器的 RNA 靶向工具

• 批准号: 10457098
• 负责人: Omar O Abudayyeh
• 金额: $ 36.3万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10457098
• 关键词: Address; Affect; Aging; Animal Model; Archaeal Genome; Bacterial Genes; Bacterial Genome; Bacterial Model; Binding; Biochemical; Biodiversity; Bioinformatics; Biological Assay; Biological Process; Biology; Biomedical Research; Biotechnology; Cell model; Cell physiology; Cells; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Codon Nucleotides; Communities; Development; Disease; Disease model; Engineering; Enterobacteria phage MS2; Enzymes; Escherichia coli; Family; Generations; Genes; Genetic Screening; Genetic Transcription; Genome; Genome engineering; Genomics; Guide RNA; Hematopoietic stem cells; Human; In Vitro; Malignant Neoplasms; Mammalian Cell; Metagenomics; Methods; Modeling; Modification; Molecular Biology; Mus; Nucleic Acid Amplification Tests; Orthologous Gene; Pathway interactions; Protein Engineering; Protein Isoforms; Proteins; RNA; RNA Binding; RNA Editing; RNA Splicing; Recording of previous events; Reporter; Resistance; Resources; Ribonucleases; Sampling; Screening procedure; Signal Transduction; Specificity; Study models; System; Technology; Testing; Toxic effect; Transcript; Work; base; cell type; design; enzyme activity; epitranscriptomics; genetic variant; genome-wide; hematopoietic stem cell aging; improved; in vitro activity; in vivo; knock-down; novel; nuclease; programs; screening; technology development; tool; transcription factor; transcriptome

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.

项目摘要 尽管基因组工程取得了非凡的进展，但精确和有效的转录组工程工具 缺乏。虽然我们和其他人已经表征了新型可编程RNA靶向CRISPR系统， 作为Cas 13，以及从这些系统开发的工具，这些工具的使用在细胞系统中受到限制， 称为附带活性的非混杂切割活性，并且Cas 13的主要应用已经是 使用用于报告信号产生的附带活性进行快速和灵敏的核酸检测。当Cas 13 已经显示在某些细胞类型中具有特异性RNA切割活性，其他细胞类型具有显著的 细胞RNA的侧切，导致细胞模型中的毒性。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仅切割靶底物。拟议的工作将涉及 这些需要通过结合计算发现，生物化学表征和酶工程， 寻找新的RNA靶向CRISPR核酸酶，使这些酶适应哺乳动物的使用，并开发特异性 用于转录组工程和转录组范围筛选的RNA靶向工具。发现和 这些新CRISPR蛋白的表征将建立在我们CRISPR酶工作的深厚历史基础上， 并借鉴新的高通量方法来挖掘生物多样性。我们会寻找 在CRISPR阵列附近富集的RNase结构域，并表征这些酶。初步 一个含有RNA酶的家族的表征，该家族含有Cas 7样RAMP RNA酶结构域，在此称为 Cas 7 -11显示了使用短向导RNA对特异性靶标的RNA切割，而没有可观察到的附带活性。 这种Cas 7 -11效应子属于III-E型系统，并且是第一个在类中表征的单蛋白效应子。 1系统。我们表征了Cas 7 -11的机制，显示了使其对肿瘤无催化活性的残基。 RNA结合应用，以及工程化Cas 7 -11用于哺乳动物细胞中的RNA敲减和编辑。使用 作为一种特异性Cas 7 -11工具，我们提出开发一种能够RNA敲低的单一技术， 基于crRNA的RNA编辑或RNA剪接，允许在单个RNA干扰中完成多个RNA干扰。 基因组范围的RNA靶向筛选，并允许有效地询问细胞回路。多 从这些发现和工程努力中产生的技术将克服现有技术的局限性， 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种多样的基因的模式。 细菌基因，通过一系列测定评估生物化学活性，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， 对基本生物过程和疾病机制进行更深入的探索。