Engineering platforms for editing RNA with single base resolution

单碱基分辨率 RNA 编辑工程平台

基本信息

批准号：
9922944
负责人：
Pablo Perez-Pinera
金额：
$ 32.01万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9922944
关键词：
Adenine Adenosine Affinity Agriculture Amino Acid Sequence Architecture Basic Science Binding Binding Proteins Biology Biotechnology Cancer Etiology Cells Chimeric Proteins Complementary RNA Complex Coupled Custom Cytidine Cytidine Deaminase DNA Data Deaminase Development Double-Stranded RNA Engineering Enzymes Family GTP-Binding Protein alpha Subunits, Gs Genes Genetic Diseases Genetic Engineering Genomic DNA Goals Human Inosine Knock-out Lead Mammalian Cell Mediating Medicine Mendelian disorder Messenger RNA Methods Modification Mutation Nonhomologous DNA End Joining Nucleic Acid Binding Nucleotides Outcome Pathway interactions Phenotype Point Mutation Protein Binding Domain Protein Engineering Proteins Protocols documentation RNA RNA Binding RNA Editing RNA Probes RNA Recognition Motif RNA Sequences RNA-Binding Proteins Reagent Research Resolution Risk Site Site-Directed Mutagenesis Specificity Technology Testing Work adenosine deaminase apoB mRNA editing catalytic subunit base biophysical properties design gene therapy genome editing innovation member novel novel strategies off-target site programs repaired synthetic biology tool tool development transcriptome

项目摘要

Precise modification of genomic DNA with gene editing tools is revolutionizing many scientific fields such as biotechnology and agriculture. Despite the rapid progress in genetic engineering, two important limitations remain that hinder widespread use of DNA editing tools in biomedicine. (1) DNA editing tools simply introduce stochastic mutations at target sites, which often lead to gene disruption. However, correction of most genetic diseases requires precise introduction of point mutations at target loci. (2) Gene editing tools frequently introduce mutations at off-target sites in genomic DNA. One alternative strategy to minimize this concern is editing RNA, which does not necessarily introduce permanent or hereditable mutations. Consequently, our long-term goal is to engineer tools for targeted modification of RNA with single base resolution in human cells. Our central hypothesis is that the Pumilio and FBF (PUF) RNA binding domain can be coupled with the cytidine deaminase APOBEC1 or the adenoside deaminase ADAR1 to introduce specific mutations at target sites within the human transcriptome. However, the development of these tools will require modification of some of the intrinsic biophysical properties of the three proteins. We will pursue our goal through three specific aims, which will test the following hypotheses: (1) Site specific mutagenesis of key residues within the PUF architecture can be used to modulate their binding affinity and enable the creation of PUFs that selectively bind unique sequences within the human transcriptome. (2) PUF-APOBEC1 fusion proteins will introduce specific user-defined sequences within the human transcriptome without off-target effects by optimizing the sequence of the linkers tethering both proteins and removing the protein-protein interaction domains in APOBEC1. (3) Heterologous RNA complementary of a target sequence can be used to modify specific adenines using PUFs in complex with ADAR1. This research is innovative because we will forward engineer proteins with distinct functions to create novel genetic engineering tools for editing RNA, a promising new approach that has not been sufficiently explored. These results will be significant because editing RNA with single base resolution will enable the development of multiple gene therapies for correction of monogenic diseases or specific point mutations causing cancer.

利用基因编辑工具对基因组 DNA 进行精确修饰正在给许多科学领域带来革命性的变化例如生物技术和农业。尽管基因工程取得了快速进展，但有两个重要的问题 DNA 编辑工具仍然存在局限性，阻碍其在生物医学领域的广泛使用。 (1)简单的DNA编辑工具在目标位点引入随机突变，这通常会导致基因破坏。然而，大多数的修正遗传疾病需要在目标位点精确引入点突变。 (2)基因编辑工具频繁使用在基因组 DNA 的脱靶位点引入突变。减少这种担忧的一种替代策略是编辑 RNA，这并不一定会引入永久性或可遗传的突变。因此，我们的长期目标是设计用于单碱基RNA靶向修饰的工具人体细胞的分辨率。我们的中心假设是 Pumilio 和 FBF (PUF) RNA 结合域可以与胞苷脱氨酶 APOBEC1 或腺苷脱氨酶 ADAR1 偶联，引入特异性人类转录组内靶位点的突变。然而，这些工具的开发需要改变这三种蛋白质的一些固有生物物理特性。我们将通过三个具体目标来实现我们的目标，这将检验以下假设： (1) 场地 PUF结构内关键残基的特异性诱变可用于调节它们的结合亲和力并能够创建选择性结合人类转录组内独特序列的 PUF。 (2) PUF-APOBEC1 融合蛋白将在人类转录组中引入特定的用户定义序列通过优化连接两种蛋白质的接头序列并去除脱靶效应 APOBEC1 中的蛋白质-蛋白质相互作用结构域。 (3) 与靶序列互补的异源RNA 可用于使用与 ADAR1 复合的 PUF 来修饰特定的腺嘌呤。这项研究具有创新性，因为我们将通过正向工程设计具有不同功能的蛋白质来创造用于编辑 RNA 的新型基因工程工具，这是一种有前途的新方法，但尚未得到充分的应用探索过。这些结果意义重大，因为用单碱基分辨率编辑 RNA 将使开发用于纠正单基因疾病或特定点突变的多种基因疗法导致癌症。