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

项目摘要 基于CRISPR的技术通过实现靶向DNA修饰改变了科学。DNA 编辑技术丰富了我们对DNA修复机制的理解， 经常被用来改进基因编辑的方法。然而，尽管取得了令人瞩目的成就， 靶向DNA修饰，RNA修复研究不足，RNA编辑工具有限。远景目标 这项提议的一个重要目的是了解控制RNA修复的分子机制， 新的RNA编辑工具，用于科学和医学。这里提出的研究整合了 协同努力开发基于CRISPR的工具，用于解剖宿主细胞接管的病毒机制， 鉴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 复制的在目标1中，我将重新利用RNA靶向CRISPR系统来删除，插入和取代 病毒RNA中的序列。我设计了CRISPR-Cas系统来删除SARS中的ORF 7a区域- CoV-2基因组和重建自然发生的突变。根据我以前的工作，我预计， ORF 7a的靶向缺失限制了病毒对宿主干扰素应答的抑制，并导致病毒复制 缺损此外，在本申请中开发的工具将用于测试新出现的病毒变体， 表型目的2研究宿主抗病毒核酸酶和宿主RNA连接酶在大肠杆菌中的拮抗活性。 SARS-CoV-2的复制和进化。目的3开发基于CRISPR的RNA捕获系统， 从复杂的混合物中提取序列特异性RNA。我将使用基于CRISPR的富集来绘制RNA SARS-CoV-2病毒RNA的修饰，并确定与种间 过渡。成功完成本申请的K99阶段将需要接受生物安全 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 RNA生物化学。总的来说，这里概述的研究和培训目标建立了一个坚实的科学 这将有助于我向独立过渡。

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.