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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.

项目总结