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Engineering of PPR base editors to repair pathogenic SNPs at the level of RNA

PPR 碱基编辑器工程可在 RNA 水平修复致病性 SNP

基本信息

批准号：
10359636
负责人：
Michael Lloyd Hayes
金额：
$ 43.8万
依托单位：
CALIFORNIA STATE UNIVERSITY LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-20 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10359636
关键词：
Affinity Amino Acids Base Sequence Binding Biochemistry Biological Assay C-terminal Catalytic Domain Cells Code Consensus Sequence Cytidine Deaminase Cytosine DNA Repair DNA Repair Pathway Data Deaminase Deamination Disease Elements End Point Assay Engineering Enzymes Fostering Future Genes Genetic Diseases Genome Goals Grant Guide RNA Health Human Hybrids In Vitro Investigation Knowledge Laboratories Lead Leigh Disease Link Mitochondria Mitochondrial DNA Modification Mutation Nucleotides Organelles Pathogenicity Pathology Plant RNA Plants Positioning Attribute Prevention Proteins RNA RNA Binding RNA Editing RNA Sequences Randomized Recombinants Research Reverse Transcriptase Polymerase Chain Reaction STEM career Series Site Specificity Structure Students Substrate Specificity System Technology Therapeutic Transcript Translations Underrepresented Minority Variant Vascular Plant base combinatorial design experimental study flexibility human disease improved in vitro Assay minority student mitochondrial genome mitochondrial membrane preference programs repaired restriction enzyme tool

项目摘要

Abstract Many pathogenic T-to-C SNPs have been identified in humans including several in the mitochondrial genome linked to Leigh syndrome. The mitochondrial genome is especially difficult to manipulate using existing gene editing technologies due to inefficient transfer of guide RNAs through the mitochondrial membranes. The organelle genomes of most land plants contain hundreds of ancient T-to-C mutations that are “repaired” by C-to-U RNA editing before translation to produce functional proteins. The sufficient editing apparatus in plants has been recently discovered to be comprised of a single protein with an RNA binding PPR tract domain and a C-terminal catalytic domain called the DYW domain. The PPR domains follow a combinatorial code where two polar amino acid positions strongly influence the ribobase recognized. Changes in the polar amino acids have been correlated with predictable changes in RNA substrate specificity making the PPR domains programable. This grant aims to reprogram plant PPR editing factors to recognize human SNPs. In the first aim of the proposal, the editing factor PPR65 will be manipulated through engineered amino acid changes in the PPR domains to target mitochondrial pathogenic SNPs. In a second aim, local sequence requirements imposed by the enzymatic domain will be investigated and DYW domain swapping experiments should identify a catalytic domain with the least sequence bias. Catalytic sequence bias could potentially limit application of repair of human SNPs and the diversity of targeted sequences in higher plants suggest such bias is not universal. Both aims seek to apply the plant RNA editing machinery to make specific base edits to improve human health. Advantages in using the plant system include the prevention of permanent off-target effects through RNA recognition by the PPR tract and a fully proteinaceous, compact structure that can theoretically be efficiently delivered to mitochondria. This project will also provide research opportunities for six under- represented minority students in biochemistry each semester. Primary research will foster excitement for biochemistry and lead to a greater equity into the backgrounds of students prepared for STEM careers.

摘要已经在人类中鉴定了许多致病性T-至-C SNP，包括在人类中的几种。线粒体基因组与利氏综合征有关线粒体基因组尤其是由于引导物的低效转移，难以使用现有的基因编辑技术进行操作 RNA穿过线粒体膜。大多数陆生植物的细胞器基因组包含数百个古老的T到C突变，这些突变在之前被C到U RNA编辑“修复”。翻译以产生功能性蛋白质。植物中足够的编辑装置已经被最近发现由具有RNA结合PPR束结构域的单一蛋白质组成和一个称为DYW结构域的C-末端催化结构域。PPR域遵循两个极性氨基酸位置强烈影响核糖基的组合密码认可.极性氨基酸的变化与蛋白质的可预测变化相关 RNA底物特异性使PPR结构域可编程。这笔赠款旨在重新规划植物PPR编辑因子来识别人类SNP。在第一个目标的建议，编辑因子PPR 65将通过PPR结构域中的工程化氨基酸改变来操纵针对线粒体致病性SNP。在第二个目标中，局部序列要求将研究由酶结构域施加的酶活性，并进行DYW结构域交换实验应该识别具有最小序列偏差的催化结构域。催化序列偏差可能潜在地限制了人类SNPs修复的应用和靶序列的多样性，高等植物表明这种偏见并不普遍。这两个目标都试图将植物RNA编辑机器进行特定的基础编辑，以改善人类健康。使用该工厂的优势系统包括通过RNA识别预防永久脱靶效应， PPR道和完全蛋白质的紧凑结构，理论上可以有效地传递到线粒体。该项目还将为六个以下的研究提供机会- 代表少数民族学生上生物化学课初步研究将促进兴奋的生物化学和导致更大的公平到学生的背景为STEM职业做好准备。