Programmable RNA-targeting tools

可编程 RNA 靶向工具

• 批准号: 9379750
• 负责人: Feng Zhang
• 金额: $ 109.57万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-17 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9379750
• 关键词: Address; Affect; Area; Binding; Biochemical; Biological; Brain; Cell physiology; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complement; Complex; DNA; DNA sequencing; Data; Development; Disease; Dissection; Elongation Factor; Engineering; Enzymes; Epigenetic Process; Essential Genes; Eukaryotic Cell; Event; Foundations; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genetic; Genetic Screening; Genetic Transcription; Genetic study; Genomic Segment; Genotype; Goals; Guide RNA; Knock-out; Libraries; Mammalian Cell; Messenger RNA; Methods; Molecular; Organism; Orthologous Gene; Phenotype; Population; Protein Isoforms; RNA; RNA Binding; RNA Interference; RNA Sequences; RNA interference screen; RNA-Binding Proteins; Regulatory Element; Reporter; Research Personnel; Ribonucleases; Science; Solid; Stimulus; Structure; System; Technology; Time; Transcript; Translations; Untranslated RNA; Up-Regulation; Variant; Visual; Work; base; cost effective; design; experimental study; flexibility; fluorophore; forward genetics; genetic approach; genome editing; genome-wide; genome-wide analysis; human disease; improved; insight; knock-down; microbial; novel; programs; reconstitution; response; scaffold; screening; sensor; small hairpin RNA; tool; tool development; transcriptome

Project Summary Functional understanding of the genetic circuits underlying complex phenotypes, including disease, will require robust, scalable tools for modulating and tracking transcriptional events. Currently, RNAi using shRNAs is the only high-throughput approach available, and this is largely limited to transcript knockdown. This project aims to develop a suite of broadly applicable tools for the interrogation of RNA based on CRISPR-Cas enzymes that target RNA in a programmable manner. Tools for transcript knockdown, translation upregulation, and transcript sensing will be developed, which, together, will enable dissection of genetic circuits in a dynamic, high-throughput manner, accelerating nearly all areas of biomedical science. The proposal is focused on four key goals: 1. Functionally and biochemically characterize RNA-targeting Cas enzymes, and then harness these enzymes for transcript editing in eukaryotic cells. Novel RNA-targeting CRISPR-Cas enzymes will be developed as platforms for transcript knockdown and translational upregulation. The availability of programmable tools for transcriptome editing in mammalian cells will provide new avenues for analyzing the effect of gene expression events and will greatly advance our ability to study specific mRNA isoforms, which is particularly important for understanding the brain. 2. Improve RNA knockdown screens. The current state-of-the-art for forward genetic screens in mammalian systems uses shRNAs, but these have significant off-target effects. RNAi screens based on programmable RNA-targeting enzymes will afford more robust, reliable data, boosting forward genetic approaches. 3. Create programmable reporter systems for transcript sensing. Catalytically inactive RNA-targeting Cas enzymes will be engineered to serve as programmable RNA-binding scaffolds that can be fused to various functional moieties (e.g., fluorophores) to develop transcript sensors. These sensors will enable dynamic, efficient tracking of transcriptional changes over extended periods of time and provide a means for isolating transcriptionally defined cell populations for further study. 4. Develop genome-wide screens to dissect genetic regulatory circuits. Genome-wide Cas9 knockout and activation screening will be combined with RNA-targeting sensors to develop high-throughput systems to identify genomic regions (coding and non-coding) that influence the expression of a target gene. These tools, which will be openly shared, will be broadly applicable across species and systems and will serve as a general framework for the expansion of the RNA-targeting toolbox. They will radically transform existing approaches for studying gene expression dynamics and exploring the significance of isoforms and non-coding transcripts.

项目摘要 对复杂表型（包括疾病）的遗传电路的功能性理解，将需要 用于调节和跟踪转录事件的强大、可扩展的工具。目前，使用shRNAs的RNAi是 只有高通量方法可用，并且这在很大程度上限于转录物敲低。该项目旨在 开发一套广泛适用的工具，用于基于CRISPR-Cas的RNA询问 以可编程方式靶向RNA的酶。用于转录、翻译的工具 上调和转录检测将被开发出来，它们将一起使基因电路的解剖成为可能。 以动态、高通量的方式，加速生物医学科学的几乎所有领域。 该提案侧重于四个关键目标： 1.功能和生物化学特性RNA靶向Cas酶，然后利用这些 真核细胞中的转录编辑酶。新的RNA靶向CRISPR-Cas酶将成为 作为转录物敲低和翻译上调的平台。的可用性 用于哺乳动物细胞中转录组编辑的可编程工具将为分析 基因表达事件的影响，并将大大提高我们研究特定mRNA亚型的能力， 这对理解大脑尤为重要。 2.改进RNA敲除筛选。目前最先进的遗传筛查技术， 哺乳动物系统使用shRNA，但这些具有显著的脱靶效应。RNAi筛选基于 可编程的RNA靶向酶将提供更强大，可靠的数据，促进向前遗传 接近。 3.创建可编程的报告系统，用于转录检测。无催化活性的RNA靶向 Cas酶将被工程化以用作可编程的RNA结合支架，其可以融合到 各种功能部分（例如，荧光团）来开发转录物传感器。这些传感器将使 动态、有效地跟踪转录变化，并提供一种方法， 用于分离转录定义的细胞群以供进一步研究。 4.发展全基因组筛选，剖析基因调控回路。全基因组Cas9敲除 激活筛选将与RNA靶向传感器相结合，以开发高通量系统 以鉴定影响靶基因表达的基因组区域（编码和非编码）。 这些工具将被公开分享，将广泛适用于不同物种和系统， 作为RNA靶向工具箱扩展的一般框架。它们将彻底改变现有的 研究基因表达动力学和探索异构体和非编码的意义的方法 成绩单