Exploration of Diverse Mobile Genetic Elements for Precision Genome Manipulation

探索用于精确基因组操作的多种移动遗传元件

• 批准号: 9345109
• 负责人: Feng Zhang
• 金额: $ 124.6万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9345109
• 关键词: BCAR1 gene; Bioinformatics; Biological Sciences; Biomedical Research; Characteristics; Complement; DNA; DNA Repair; Development; Disease; Enzymes; Event; Foundations; Genetic study; Genome; Genome engineering; In Vitro; Methods; Microbe; Microbial Genetics; Mobile Genetic Elements; Operative Surgical Procedures; Point Mutation; RNA; Research; Safety; Site; Specificity; Subcategory; System; Technology; Transposase; Work; Zinc Fingers; base; design; fighting; genetic element; genome editing; human disease; in vivo; innovation; insertion/deletion mutation; novel; nuclease; operation; recombinase; repaired; therapeutic gene; tool; transcription activator-like effector nucleases

The past five years have seen extraordinary advances in genome engineering technologies, yet we still lack efficient, robust tools for precise genome manipulation, particularly the ability to insert a desired sequence at a target site. Such tools would revolutionize our approach to treating human diseases and significantly increase the pace of functional genetic studies throughout the biological sciences. Current approaches are focused on the development of nucleases, such as TALENs, zinc-fingers, and Cas proteins, but there is a limit to the utility of these enzymes and they all rely on the host endogenous DNA repair machinery. Moreover, the use of a nuclease to achieve therapeutic gene editing requires additional efforts to safeguard against off-target mutagenic events. A partial solution to this challenge already exists in the form of mobile genetic elements (MGEs), systems that microbes have been using (or fighting) for millennia to achieve precise genome manipulation. We will explore the diverse pool of MGEs and identify those with characteristics that can be leveraged for eukaryotic genome editing. Precision genome manipulation can be broadly divided into three subcategories: insertions, deletions, and rearrangements, each of which forms the foundation for an array of applications. Our approach will be to consider each of these modes of operation separately to first select candidate MGEs or components of these systems through bioinformatics analysis and extrapolating from known systems. For example, transposases and recombinases may be harnessed for insertions, whereas the RNA-templated genome unscrambling that occurs in the ciliates may be suitable for adaptation for genome rearrangements. Once candidates for these three modes have been identified, we will functionally characterize them in vitro and then develop them for genome editing. Finally, we will assess the specificity, efficacy, and safety of these novel precision genome editors in vivo. This approach represents an innovative new direction in the development of genome engineering technology that overcomes the reliance on nucleases by designing new, self-contained enzymatic tools that can accomplish all three operational modes of genome manipulation. This work will complement our on-going research efforts to develop effective delivery methods for genome engineering components. Together, these technologies will allow us to realize the full potential of genome engineering, particularly as an avenue for treatment of human diseases.

在过去的五年中，基因组工程技术取得了非凡的进步，但我们仍然缺乏 有效，健壮的工具用于精确的基因组操纵，尤其是在A插入所需序列的能力 目标站点。这样的工具将彻底改变我们治疗人类疾病的方法，并大大增加 在整个生物科学中的功能遗传研究的速度。当前的方法专注于 核酸酶的开发，例如talens，锌芬和CAS蛋白，但这些效用有限制 酶和它们都依靠宿主内源性DNA修复机制。此外，使用核酸酶 实现治疗基因编辑需要额外的努力来保护脱靶诱变事件。一个 针对这一挑战的部分解决方案已经以移动遗传因素（MGE）的形式存在 微生物一直在使用（或战斗）数千年来实现精确的基因组操纵。我们将探索 各种MGE的池，并识别具有可用于真核基因组编辑的特征的人。 精确的基因组操纵可以大致分为三个子类别：插入，缺失和 重新安排，每种都为一系列应用程序构成基础。我们的方法是考虑 这些操作模式分别以首先选择这些系统的候选MGE或组件 通过生物信息学分析和从已知系统推断。例如，转座酶和 重物组织酶可以插入插入 纤毛可能适合适应基因组重排。一旦候选这三种模式 已经确定，我们将在体外功能表征它们，然后开发它们进行基因组编辑。 最后，我们将评估这些新型精确基因组编辑器体内的特异性，功效和安全性。这 方法代表了基因组工程技术发展的创新新方向 通过设计新的，独立的酶促工具来克服对核酸酶的依赖，这些工具可以完成这三个工具 基因组操纵的操作模式。这项工作将补充我们正在进行的研究工作以发展 基因组工程组件的有效输送方法。这些技术一起将使我们能够 意识到基因组工程的全部潜力，尤其是作为治疗人类疾病的途径。