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

项目总结 基于CRISPR的技术通过实现有针对性的DNA修改改变了科学。脱氧核糖核酸 编辑技术丰富了我们对DNA修复的机械理解，并对修复有了新的见解 经常用于改进基因编辑的方法。然而，尽管取得了令人瞩目的成就， 有针对性的DNA修饰、RNA修复研究不足，RNA编辑工具有限。长期目标 这一建议的目的是了解支配RNA修复的分子机制，并开发破坏性的 新的RNA编辑工具在科学和医学中的应用。这里提出的研究整合了 协同努力开发基于CRISPR的工具，用于剖析宿主细胞接管和 识别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，以删除、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，并重新创造自然发生的突变。基于我之前的工作，我预计 ORF7a中的靶向缺失限制了宿主干扰素反应的病毒抑制并导致复制 叛逃。此外，在此应用程序中开发的工具将用于测试新出现的病毒变体 表型。目的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的复制和进化。Aim 3开发了一个基于CRISPR的RNA捕获系统来丰富 从复杂混合物中提取特定序列的RNA。我将使用基于CRISPR的富集化来映射RNA SARS-CoV-2病毒RNA的修饰和鉴定与种间相关的分子特征 过渡。成功完成这项申请的K99阶段将需要在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的能力 在RNA生物化学方面。总的来说，这里概述的研究和培训目标建立了坚实的科学 这将促进我向独立的过渡。

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.