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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：
10505965
负责人：
Artem Aleksandrovich Nemudryi
金额：
$ 13.82万
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/10505965
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 Lead Lesion Ligase 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 Research Training Ribonucleases Role SARS-CoV-2 genome Sampling Science Shapes Site-Directed Mutagenesis Solid Syndrome System Tail Technology Testing Training 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 ribonuclease E 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.

项目总结