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

摘要 已在人类中发现了许多致病的T-to-C单核苷酸多态，其中包括几个 线粒体基因组与Leigh综合征有关。尤其是线粒体基因组 由于引导转移的低效，使用现有的基因编辑技术难以操作 通过线粒体膜的RNA。大多数陆地植物的细胞器基因组 包含数百个古老的T-to-C突变，这些突变在以前的C-to-U RNA编辑过程中被“修复” 翻译以产生功能蛋白质。植物中充足的编辑设备已经被 最近发现由一个带有RNA结合PPR区域的单一蛋白质组成 以及一个C端催化结构域，称为dyw结构域。PPR域遵循 两个极性氨基酸位置对核糖基有强烈影响的组合编码 被认可了。极性氨基酸的变化与可预测的 RNA底物专一性使PPR结构域可编程。这笔拨款的目的是重新编程 植物PPR编辑因子识别人类SNPs。在提案的第一个目标中，编辑 因子PPR65将通过PPR结构域中的工程氨基酸变化来操纵 以线粒体致病SNPs为靶点。在第二个目标中，本地序列要求 所施加的酶结构域将被研究并进行dyw结构域交换实验 应该确定一个序列偏差最小的催化结构域。催化序列偏差可能 潜在地限制了人类SNPs修复的应用和靶向序列的多样性 高等植物表明，这种偏见并不普遍。这两个目标都寻求应用植物RNA编辑 机器进行特定的基本编辑，以改善人类健康。使用该植物的优势 系统包括通过RNA识别来防止永久性的脱靶效应 和一种完全含蛋白质的紧凑结构，理论上可以有效地 传送到线粒体。该项目还将为六名以下儿童提供研究机会。 每学期代表少数族裔学生学习生物化学。初步研究将促进 对生物化学的热情和对学生背景的更大公平 为STEM职业生涯做好准备。

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.