Advancing programmable RNA-targeting tools for research and therapeutics

推进用于研究和治疗的可编程 RNA 靶向工具

基本信息

批准号：
10273820
负责人：
Feng Zhang
金额：
$ 78.4万
依托单位：
BROAD INSTITUTE, INC.
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-17 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10273820
关键词：
Acute Adopted Binding Biochemical Bioinformatics Biological Sciences Biology Brain Cell Line Cells Clinic Clinical Clustered Regularly Interspaced Short Palindromic Repeats DNA Deaminase Development Disease Disease model Engineering Enzymes Evolution Future Genetic Diseases Genetic Transcription Goals Human Biology Image Kinetics Light Mammalian Cell Mining Modification Molecular Molecular Biology Molecular Conformation Mus Mutation Neurodevelopmental Disorder Noise Orthologous Gene Population Protein Engineering Proteins RNA RNA Editing RNA Sequences RNA Stability RNA-Binding Proteins Recovery Research Research Personnel Rett Syndrome Role Route Signal Transduction Specificity Structure System Technology Testing Therapeutic Therapeutic Uses Tissues Transcript Variant Viral Vector Work base cell type computational pipelines experience functional outcomes gene therapy genome editing human disease imaging platform improved in vivo knock-down liver injury microbial mouse model neurodevelopment novel novel strategies preference programs reconstitution sensor technology development therapeutic genome editing tool transcriptome

项目摘要

PROJECT SUMMARY We previously developed a suite of tools for modulating and studying RNA based on the RNA-targeting CRISPR- Cas13 system, which has been adopted or extended by many researchers in the life sciences. This proposal seeks to discover and characterize additional programmable RNA binding proteins and develop them for use as molecular technologies. In particular, we are focusing on identifying ultra-small Cas13 proteins, which can be fused to RNA editing effectors to create compact platforms for precision, single-base transcript editing. RNA editing has significant therapeutic potential across a spectrum of conditions, including genetic diseases where it is not possible or too risky to edit the genome as well as acute insults where transient genetic changes are desirable. Thus, another aim is to demonstrate the feasibility of using RNA editing therapeutically. Beyond RNA editors, we will also use the new proteins we identify to develop transcriptional state sensors, which can be used to mark specific cell sub-types within a heterogenous population, either for imaging, isolation, or functional outcomes. To achieve these goals, we will leverage our previous experience to discover and characterize new RNA targeting CRISPR systems, with a focus on identifying small enzymes that support RNA editing activity. We will also explore the possibility of using novel RNA deaminase enzymes in our RNA editing constructs. In addition to creating RNA editing constructs, we will also fuse the RNA targeting enzymes to GFP or Cre to create transcriptional sensors. A critical aspect of our work will be protein engineering. Candidate enzymes (or their RNA components) may need to be modified for efficient, specific activity in mammalian cells. We will use protein engineering to increase the specificity and activity of RNA deaminases, as well as extend the substrate base preference of these enzymes. For our transcriptional state sensors, protein engineering will be central to successfully generating sensors that afford high specificity and high signal-to-noise ratios. All of these efforts will be guided by structural and biochemical studies of the relevant enzymes. Finally, we will apply the compact, high-specificity RNA editors in a mouse model of acute liver damage to demonstrate their therapeutic potential as short-lived, reversible treatments. In parallel, we will demonstrate the feasibility of using RNA editing to correct a mutation that causes the neurodevelopmental disorder Rett syndrome using a previously established mouse model of this disease. This work will substantially advance RNA editing toward clinical use, as well as uncover new biology about CRISPR systems and other microbial defense systems.

项目摘要我们之前开发了一套工具，用于根据RNA靶向CRISPR-调节和研究RNA CAS13系统已被生命科学中的许多研究人员采用或扩展。这个建议试图发现和表征其他可编程RNA结合蛋白并开发它们用作分子技术。特别是，我们专注于识别超小的CAS13蛋白，可以将其融合到RNA编辑效应子中，以创建紧凑的平台，以进行精确的单基数成绩单编辑。 RNA编辑在各种条件下具有明显的治疗潜力，包括遗传疾病不可能或太冒险编辑基因组以及急性侮辱是短暂的遗传更改是可取的。因此，另一个目的是证明使用RNA进行治疗的可行性。除了RNA编辑器之外，我们还将使用我们识别的新蛋白质来开发转录状态传感器，这些传感器可用于在异源种群中标记特定的细胞亚型，以成像，分离或功能结果。为了实现这些目标，我们将利用我们以前的经验来发现和表征新的RNA 针对CRISPR系统，重点是识别支持RNA编辑活性的小型酶。我们将还可以探索在我们的RNA编辑构建体中使用新型RNA脱氨酶的可能性。此外创建RNA编辑结构，我们还将融合RNA靶向酶与GFP或CRE创建转录传感器。我们工作的一个关键方面将是蛋白质工程。候选酶（或他们的 RNA成分）可能需要修改以在哺乳动物细胞中有效，特异性活性。我们将使用蛋白质工程以提高RNA脱氨酶的特异性和活性，并扩展底物基础这些酶的偏爱。对于我们的转录状态传感器，蛋白质工程将是成功地生成具有高特异性和高信噪比的传感器。所有这些努力将以相关酶的结构和生化研究为指导。最后，我们将在急性肝损伤的小鼠模型中应用紧凑的高特异性RNA编辑器证明其治疗潜力是短暂的，可逆的治疗方法。同时，我们将展示使用RNA编辑来纠正引起神经发育障碍RETT综合征的突变的可行性使用先前建立的该疾病的小鼠模型。这项工作将大大推动RNA编辑临床使用，并发现有关的新生物学 CRISPR系统和其他微生物防御系统。