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.

使用基因编辑工具对基因组DNA进行精确修饰正在彻底改变许多科学领域 例如生物技术和农业。尽管基因工程进展迅速， 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 在靶位点引入随机突变，这通常导致基因破坏。然而，大多数修正 遗传疾病需要在靶基因座精确引入点突变。(2)基因编辑工具经常 在基因组DNA的脱靶位点引入突变。一种将这种担忧降到最低的替代策略是 编辑RNA，这不一定会引入永久性或遗传性突变。 因此，我们的长期目标是设计单碱基靶向修饰RNA的工具 在人体细胞中的分辨率。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APOBEC 1或腺苷脱氨酶ADAR 1偶联，以引入特异性 人类转录组内靶位点的突变。然而，这些工具的开发将需要 修饰这三种蛋白质的一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，这些目标将检验以下假设：（1）地点 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 并且能够产生选择性结合人转录组内的独特序列的PUF。（二） PUF-APOBEC 1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的序列并去除 APOBEC 1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ADAR 1复合的PUF修饰特定腺嘌呤。 这项研究是创新的，因为我们将提出具有不同功能的工程蛋白质， 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，但还没有得到充分的研究。 探讨了这些结果将是重要的，因为用单碱基分辨率编辑RNA将使得能够进行RNA的测序。 开发用于校正单基因疾病或特定点突变的多种基因疗法 导致癌症。