Building the mitochondrial genome editing repertoire

摘要 线粒体在新陈代谢、器官动态平衡、细胞凋亡和衰老等方面具有重要的正常作用。线粒体 在人类生理学中扮演着重要但在很大程度上仍然神秘的角色。两个核DNA相关的突变 随着蛋白质输入线粒体，线粒体DNA(MtDNA)是致病的。尽管如此 明确了基因与疾病的关联，目前还没有治疗线粒体疾病的方法 疾病。 线粒体代表着一个独特的细胞隔间，具有不同的DNA和RNA修复和编辑 规矩。例如，在核DNA中引入双链断裂和随后修复的DNA核酸酶 诱导线粒体DNA降解。事实上，在细胞核中发现的常见修复途径中没有一条是 活跃在线粒体中。 该方案使用成熟的基于TALL的可编程DNA结合系统来靶向 线粒体DNA和单管FUSX TALL组装系统快速生成任何基于蛋白质的基因组 工程试剂。同样，我们使用一种新的、基于蛋白质和可编程的RNA结合系统 在PPR蛋白上，一类来自植物的自然产生的、线粒体定位的RNA编辑程序。 这个应用程序利用线粒体的独特环境生成一个新的工具箱 扩展编辑人类基因组的工具库(RFA-RM-18-017)。开发这些新的 作为mtDNA和mtRNA体细胞编辑的分子试剂，我们将进行以下目标： 开发新类别的线粒体DNA编辑工具。改进了使用MitoTALEN的方法 将开发MELAS和KSS的致病mtDNA变异的优先降解，包括 新的核编码报告，用于检测非线粒体脱靶基因编辑事件。一种新的 将开发一类TALE线粒体碱基编辑程序，用于直接编辑致病变种的mtDNA。 二.线粒体RNA编辑工具将通过利用自然发生的PPR家族产生 可编程的RNA编辑器。我们将使用我们新的FUSR组装系统来快速开发最佳RNA 结合试剂，包括融合到一组测试RNA核酸酶或编辑蛋白结构域。错误的 MtDNA缺失的融合转录本或异质细胞中的单碱基变异将被用作测试 潜在的RNA编辑平台开发的范例，具有潜在的治疗作用。 最初阶段的里程碑包括建立新的线粒体DNA异质性转化有丝分裂标记 对MELAS和KSS进行了比对，然后测试了新的mtDNA碱基编辑器。对于mtRNA编辑，建立 MtRNA结合的PPR支架规则以及新的可编程RNA核酸酶和编辑器将 已经成立了。交付成果包括这些新的mtDNA和mtRNA编辑系统以及人类细胞 具有匹配的核编码的脱靶报告盒，可用于任何线粒体基因编辑 治疗系统。

Abstract Mitochondria have critical normal roles in metabolism, organ homeostasis, apoptosis and aging. Mitochondria play important but still largely mysterious roles in human physiology. Mutations in both nuclear DNA associated with proteins imported into mitochondria as well as mitochondrial DNA (mtDNA) are pathogenic. Despite this clear association of genotype with disease, there are no current treatments for patients with mitochondrial disease. Mitochondria represent a unique cellular compartment with different DNA and RNA repair and editing rules. For example, DNA nucleases that introduce double strand breaks and subsequent repair in nuclear DNA induce the degradation of mtDNA. Indeed, none of the common repair pathways found in the nucleus are active in mitochondria. This proposal uses the well-established TALE-based programmable DNA binding system for targeting mtDNA and the single-tube FUSX TALE assembly system to rapidly generate any protein-based genome engineering reagents. Similarly, we use a new, protein-based and programmable RNA binding system based on PPR proteins, a class of naturally occurring, mitochondrially localized RNA editors from plants. This application harnesses the unique environment of mitochondria to generate a new toolbox to expand the repertoire of tools to edit the human genome (RFA-RM-18-017). To develop these new molecular reagents for mtDNA and mtRNA editing of somatic cells, we will conduct the following aims: I. Develop new classes of mtDNA editing tools. Enhanced approaches to the use of mitoTALENs for preferential degradation of pathogenic mtDNA variants for MELAS and KSS will be developed, including novel nuclearly encoded reporters to detect non-mitochondrial off-targeting gene editing events. A new class of TALE mitochondrial base editors will be developed to directly edit mtDNA for pathogenic variants. II. mtRNA editing tools will be generated through harnessing the PPR family of naturally occurring programmable RNA editors. We will use our new FUSR assembly system to rapidly develop optimal RNA binding reagents, including the fusion to a set of test RNA nuclease or editing protein domains. Errant fusion transcripts in mtDNA deletion or single base variants in heteroplasmic cells will be used as the test paradigm for potential RNA editing platform development with the potential use as a therapeutic. Milestones for initial stages include the establishment of novel mtDNA heteroplasmy converting mitoTALENs against MELAS and KSS followed by testing of the new mtDNA base editor. For mtRNA editing, establishing PPR scaffolding rules for mtRNA binding followed by new programmable RNA nucleases and editors will be established. Deliverables include these novel mtDNA and mtRNA editing systems as well as humans cells with matched nuclearly encoded off-target reporter cassettes for use by any mitochondrial gene editing therapeutic system.