基于转录激活因子样效应因子(TALEs)的人类线粒体基因组编辑技术体系的构建与相关基础研究

Mitochondia (Mt) dysfunction due to mtDNA mutations can lead to inherited mitochondiral and aging diseases in humans. However, there is no effective way to rectify mitochondrial abnormalities mainly due to the lack of aproach to manipulate or modify particular mtDNA sequences in mitochondria within cells.Compared to zinc finger proteins technology (ZFPs),transcription activator like effectors (TALEs)technique is a recently emerged and more powerful technology used for targeting and modifying the specific sequence in human genome.In this study, a novel TALEs system specifically targeting the mitochondrial genome sequence (mitochondrial transcription activator-like effector nuclease,mtTALEN) has be established. This system mainly consists of two parts:mtTALEs backbones and central tandem repeat domain (CRD)assembly system.In the first,the N terminal nuclear localization signal(NLS) from a common TALEN backbone was replaced with a strong nuclear export signal(NES) and a mitochondrial targeting sequence (MTS)from homo sapiens ornithine carbamoyltransferase (OTC). In the second, A bias-unit library which contains 92 plasmids (12 single units,16 double units and 64 triple units) has been constructed using XbaI-NheI isocaudamer strategy, which are the base modules for assemblying CRD in a normal or high-throughput way.Based on this system, our preliminary data have clearly demonstrated the mitochondrial localization of mtTALENs fusion protein by immunofluorescence staining and western blotting in U2OS cells.In this study, we will test the selective degradation of mutated mtDNA by mtTALENs in heteroplasmic cells which containing a mixture of mutated and wildtype mtDNA in aim to clarify the poteintial use of mtTALENs system in the treatment of mtDNA diseases.In addition, mtTALENs will be used as a cutting tools to generate precise double strand breaks in mtDNA in aim to define the molecular mechanism of mtDNA damage repair.

mtDNA突变是导致人类线粒体遗传病及衰老性疾病的重要原因。目前由于缺乏对mtDNA特殊序列进行改造的能力，对这类疾病仍无有效治疗手段。TALEs是继锌指蛋白(ZFPs)之后发展出的一种新型基因组靶向编辑修饰技术。本研究构建了独立的mtTALEs系统，由两部分组成：mtTALEs骨架和中央重复单元(CRD)组装系统。1)将普通TALEs骨架NLS删除，取代以HIV-rev-NES和OTC蛋白MTS，得到mtTALEs骨架，成功表达并验证其线粒体定位。2)利用同尾酶XbaI-NheI偏单元策略，构建了包含92个质粒的一、二、三单元库作为普通或高通量组装CRD的基础模块。我们还将测试mtTALEN在mtDNA异质性细胞中选择性降解突变mtDNA的能力，以验证其作为潜在线粒体病治疗手段的可能性；并将以mtTALEN作为剪切工具，以期深入探讨H2AX等核蛋白参与mtDNA双链断裂修复的机制。

mtDNA 突变是导致人类线粒体遗传病及衰老性疾病的重要原因。目前由于缺乏对 mtDNA 特殊序列进行改造的能力，对这类疾病仍无有效治疗手段。TALEs及CRISPR/Cas9是继锌指蛋白（ZFPs）之后发展的两种新型基因组靶向编辑修饰技术。本研究在不影响中央重复单元(CRD)专一性与亲和力的情况下，优化了TALENs模块组装方法。利用XbaI-NheI偏单元策略，构建了包含一系列一、二和三单元库的基础单元库，使得模块具有多样性，同时也提高了组装效率及蛋白表达效率。另外，我们构建了独特的靶向线粒体DNA的mtTALEs系统。将TALENs的核定位信号序列（nuclear localization signal，NLS）替换为线粒体前导肽序列（mitochondrial targeting sequence，MTS），命名为MTS-mtTALENs，并添加了出核信号肽序列 (nuclear export signal，NES)，命名为MTS+NES-mtTALENs，利用细胞免疫荧光技术和分离线粒体，检测 mtTALENs 与线粒体共定位及在线粒体内的表达量情况，结果显示MTS+NES-mtTALENs和MTS-mtTALENs均可以定位于线粒体。活性检测结果表明，MTS+NES-mtTALENs剪切效率较高，且对核基因组干扰较小。并且，我们还对mtDNA 双链断裂修复的机制进行了探索性研究，首次证明了线粒体DNA不存在非同源末端连接介导的DNA双链断裂修复机制，但该结论仍需进一步证实。除此之外，本研究还构建了靶向线粒体基因组的mtCRISPR/Cas9 系统以及建立了一种检测线粒体核酸酶靶向剪切活性的新方法。