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.

抽象的在人类中已经确定了许多致病性T-to-c SNP 线粒体基因组与Leigh综合征有关。线粒体基因组尤其是由于指南的效率低下，使用现有基因编辑技术难以操纵 RNA通过线粒体膜。大多数土地植物的细胞器基因组包含数百个古老的T-t-c-c突变，这些突变已通过C-TO-U RNA编辑“修复” 翻译以产生功能蛋白。植物中足够的编辑设备已经最近被发现已完成为具有RNA结合PPR域的单个蛋白质和一个称为DYW结构域的C末端催化结构域。 PPR域遵循组合代码，其中两个极性氨基酸位置强烈影响核对酶认可。极性氨基酸的变化与可预测的变化有关 RNA底物特异性使PPR域可编程。该赠款旨在重新编程植物PPR编辑因素识别人类SNP。在提案的第一个目的中，编辑因子PPR65将通过PPR域中的工程氨基酸变化来操纵靶向线粒体致病性SNP。在第二个目标中，本地序列要求将研究酶结构域施加的，并进行DYW域交换实验应鉴定具有最小序列偏差的催化域。催化序列偏差可能潜在地限制了人类SNP修复的应用以及目标序列的多样性较高的植物表明这种偏见不是普遍的。两种目的都试图应用植物RNA编辑进行特定基础编辑以改善人类健康的机械。使用植物的优势系统包括通过通过RNA识别的RNA识别的永久脱靶效应 PPR道和完全蛋白质的紧凑结构，理论上可以有效传递到线粒体。该项目还将为六个不足的研究机会提供研究机会每个学期都代表少数族裔学生的生物化学。基本研究将促进对生物化学的兴奋，并导致更大的平等进入学生的背景为STEM职业准备。