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

基本信息

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
项目类别：
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

项目摘要

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方面取得了非凡的进步，但用于精确有效的转录组工程的工具缺乏。尽管我们和其他人都表征了新型可编程RNA靶向CRISPR系统，但作为CAS13和这些系统中开发的工具，这些工具的使用在蜂窝系统中受到限制对于称为附带活动的非司法切割活动，CAS13的主要应用是使用附带活性来生成记者信号的快速和敏感的核酸测试。而cas13 已显示在某些细胞类型中具有特定的RNA裂解活性，其他细胞类型具有显着细胞RNA的侧支切割，导致细胞模型的毒性。哺乳动物的理想RNA靶向工具应用将缺乏抵押活动，并且只能切割目标底物。拟议的工作将解决通过结合计算发现，生化表征和酶工程的这些需求找到靶向CRISPR核酸核酸酶的新的RNA，适应这些酶用于哺乳动物，并开发特定 RNA定位工具用于转录组工程和整个转录组筛选。发现和这些新的CRISPR蛋白的表征都将基于我们CRISPR酶工作的深刻历史，以及从新的高通量方法中抽出矿山生物学多样性的方法。我们将寻找家庭 RNase域富含CRISPR阵列，并表征这些酶。初步的一个含有cas7样的坡道RNase域的一个含有一个RNase的表征，此处称为 CAS7-11，使用短导RNA显示了特定靶标的RNA裂解，而无需观察到侧支活性。该CAS7-11效应器属于III-E型系统，是类中第一个特征的单蛋白效应器 1系统。我们表征了CAS7-11的机制，显示了使其催化无活性的残基 RNA结合应用和哺乳动物细胞中RNA敲低和编辑的工程师CAS7-11。使用特定的CAS7-11工具，我们建议开发一种能够敲低RNA的单个技术基于CRRNA的编辑或RNA剪接，允许在单个中完成多个RNA扰动基因组宽的RNA靶向筛选，并有效询问细胞回路。倍数这些发现和工程工作产生的技术将克服现有的局限性转录组工程的方法，并成为更广泛的生物医学研究的宝贵资源。此外，这个基因探索和工程框架将成为发现多样的模型细菌基因，评估一系列测定范围的生化活性，并将这些发现转化为高影响生物技术。开发的技术将加速生物医学研究的步伐，并启用对基本生物学过程和疾病机制的更多探索。