Engineering platforms for editing RNA with single base resolution

单碱基分辨率 RNA 编辑工程平台

• 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
• 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

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.

用基因编辑工具精确地修改基因组DNA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，但两个重要的 限制仍然存在，阻碍了DNA编辑工具在生物医学中的广泛使用。(1)DNA编辑工具简单 在目标位置引入随机突变，这往往会导致基因中断。然而，大多数人的纠正 遗传性疾病需要在目标基因座精确引入点突变。(2)频繁使用基因编辑工具 在基因组DNA中的非靶点位置引入突变。将这种担忧降至最低的一种替代策略是 编辑RNA，这不一定会带来永久性或可遗传的突变。 因此，我们的长期目标是设计具有单一碱基的RNA靶向修饰工具 在人类细胞中的分辨率。我们的中心假设是，Pumilio和FbF(PUF)RNA结合结构域可以 与胞苷脱氨酶APOBEC1或腺苷脱氨酶ADAR1偶联以引入特异性 人类转录组中靶点的突变。然而，这些工具的开发将需要 修饰这三种蛋白质的某些固有生物物理性质。 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)站点 PUF结构内关键残基的特异性突变可用于调节它们的结合亲和力 并且能够产生选择性地结合人类转录组内的独特序列的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 通过优化连接这两种蛋白质的连接体的序列并去除 APOBEC1中的蛋白质-蛋白质相互作用结构域。(3)靶序列的异源RNA互补 可用于使用与ADAR1形成的络合物中的PUF来修饰特定的腺嘌呤。 这项研究具有创新性，因为我们将转发具有不同功能的工程蛋白质来创造 编辑RNA的新基因工程工具，一种前景看好但还不够充分的新方法 探索过了。这些结果将具有重要意义，因为编辑具有单一碱基分辨率的RNA将使 用于纠正单基因疾病或特定点突变的多基因疗法的发展 致癌。