CRISPR-based editing of RNA and cellular mechanisms that govern RNA repair

基于 CRISPR 的 RNA 编辑和控制 RNA 修复的细胞机制

基本信息

批准号：
10685479
负责人：
Artem Aleksandrovich Nemudryi
金额：
$ 13.77万
依托单位：
MONTANA STATE UNIVERSITY - BOZEMAN
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-17 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10685479
关键词：
2019-nCoV Achievement Acute BCAR1 gene Bioinformatics Biological Biological Assay Biotechnology COVID-19 detection CRISPR/Cas technology Cell Line Cells Clinical Clustered Regularly Interspaced Short Palindromic Repeats Competence Complex Complex Mixtures Conflict (Psychology)Coronavirus DNA DNA Repair Data Defect Detection Diagnostic Epigenetic Process Evolution Foundations Gene Expression Regulation Genes Genetic Genome Goals Guide RNA Human Impairment In Vitro Interferons Laboratories Lesion Ligase Ligation Maps Mass Spectrum Analysis Measures Mediating Medicine Mentors Messenger RNA Metabolism Methods Modification Molecular Molecular Profiling Mutation Nature Outcome Pattern Phase Phenotype Play Positioning Attribute Process Proteins Publishing RNA RNA Biochemistry RNA Editing RNA Ligase (ATP)RNA Sequences RNA Splicing RNA Viruses RNA metabolism Research Research Proposals Ribonucleases Role SARS-CoV-2 genome Sampling Science Shapes Site-Directed Mutagenesis Solid Syndrome System Tail Technology Testing Training Transfection Transfer RNA Variant Viral Viral Proteins Virus Virus Replication Work base base editing biosafety level 3 facility career deep sequencing design fitness genome integrity genomic RNA improved insight knock-down mutant nuclease overexpression recruit repaired respiratory response tRNA Ligase tool transcriptome transcriptome sequencing viral RNA

项目摘要

PROJECT SUMMARY CRISPR-based technologies have transformed science by enabling targeted DNA modification. DNA editing technologies have enriched our mechanistic understanding of DNA repair, and new insights in repair are frequently used to improve methods for gene editing. However, despite the remarkable achievements in targeted DNA modifications, RNA repair is understudied, and RNA editing tools are limited. The long-term goal of this proposal is to understand the molecular mechanisms that govern RNA repair and to develop disruptive new RNA editing tools for applications in science and medicine. The research proposed here integrates synergistic efforts to develop CRISPR-based tools for dissecting viral mechanisms of host cell take over and identify viral RNA repair mechanisms that support acute respiratory syndrome coronavirus (SARS-CoV-2) replication. In Aim 1, I will repurpose RNA-targeting CRISPR systems for deleting, inserting, and substituting sequences in viral RNAs. I have designed CRISPR-Cas systems to delete regions of ORF7a in the SARS- CoV-2 genome and recreate naturally occurring mutations. Based on my previous work, I anticipate that targeted deletions in ORF7a limit viral suppression of the host interferon response and lead to a replication defect. Further, tools developed in this application will be used to test emerging viral variants for new phenotypes. Aim 2 investigates the antagonistic activities of host antiviral nucleases and host RNA ligases in the replication and evolution of SARS-CoV-2. Aim 3 develops a CRISPR-based RNA capture system to enrich sequence-specific RNAs from a complex mixture. I will use CRISPR-based enrichment to map RNA modifications in SARS-CoV-2 viral RNAs and identify molecular signatures associated with interspecies transitions. Successful completion of the K99 phase of this application will require training in the Biosafety Level 3 facility, bioinformatics training to identify and annotate CRISPR systems, as well as new competencies in RNA biochemistry. Collectively, the research and training objectives outlined here establish a solid scientific foundation that will facilitate my transition to independence.

项目摘要基于CRISPR的技术通过实现靶向DNA修饰来改变科学。脱氧核糖核酸编辑技术丰富了我们对DNA修复的机械理解和新的修复见解经常用于改善基因编辑方法。然而，尽管取得了显着的成就靶向DNA修饰，RNA修复被研究了，RNA编辑工具受到限制。长期目标该建议的是了解控制RNA修复并发展破坏性的分子机制新的RNA编辑工具用于科学和医学中的应用。这里提出的研究集成了开发基于CRISPR的工具来剖析宿主细胞的病毒机制的协同努力接管和确定支持急性呼吸综合征冠状病毒（SARS-COV-2）的病毒RNA修复机制复制。在AIM 1中，我将重新利用RNA的CRISPR系统来删除，插入和替换病毒RNA中的序列。我设计了CRISPR-CAS系统来删除SARS中的ORF7A区域 COV-2基因组并重现天然发生的突变。根据我以前的工作，我期望 ORF7A中有针对性的缺失限制宿主干扰素反应的病毒抑制，并导致复制缺点。此外，该应用程序中开发的工具将用于测试新的病毒变体表型。 AIM 2研究宿主抗病毒核酸酶和宿主RNA连接酶的拮抗活性 SARS-COV-2的复制和演变。 AIM 3开发了基于CRISPR的RNA捕获系统以丰富来自复杂混合物的序列特异性RNA。我将使用基于CRISPR的富集来映射RNA SARS-COV-2病毒RNA的修饰并确定与种间有关的分子特征过渡。成功完成本申请的K99阶段将需要在生物安全中进行培训 3级设施，生物信息学培训以识别和注释CRISPR系统以及新的能力在RNA生物化学中。总的来说，这里概述的研究和培训目标建立了一个扎实的科学将有助于我向独立过渡的基础。