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.

项目概要基于 CRISPR 的技术通过实现定向 DNA 修饰改变了科学。脱氧核糖核酸编辑技术丰富了我们对DNA修复机制的理解，并对修复有了新的认识经常用于改进基因编辑方法。然而，尽管取得了令人瞩目的成就靶向 DNA 修饰、RNA 修复尚未得到充分研究，RNA 编辑工具也很有限。长期目标该提案的目的是了解控制 RNA 修复的分子机制并开发破坏性的用于科学和医学应用的新型 RNA 编辑工具。这里提出的研究整合了协同努力开发基于 CRISPR 的工具来剖析宿主细胞接管和病毒机制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。在目标 1 中，我将重新调整 RNA 靶向 CRISPR 系统的用途，用于删除、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 基因组并重建自然发生的突变。根据我之前的工作，我预计 ORF7a 中的靶向删除限制了病毒对宿主干扰素反应的抑制并导致复制缺点。此外，本应用中开发的工具将用于测试新出现的病毒变种表型。目标 2 研究宿主抗病毒核酸酶和宿主 RNA 连接酶在 SARS-CoV-2 的复制和进化。 Aim 3 开发基于 CRISPR 的 RNA 捕获系统来富集来自复杂混合物的序列特异性RNA。我将使用基于 CRISPR 的富集来绘制 RNA 图谱 SARS-CoV-2 病毒 RNA 的修饰并鉴定与种间相关的分子特征过渡。成功完成本申请的 K99 阶段将需要生物安全方面的培训 3 级设施、识别和注释 CRISPR 系统的生物信息学培训以及新能力在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坚实的科学基础基金会将有助于我过渡到独立。