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.

过去五年，基因组工程技术取得了非凡的进步，但我们仍然缺乏 用于精确基因组操作的高效、稳健的工具，特别是在基因组中插入所需序列的能力。 目标部位。这些工具将彻底改变我们治疗人类疾病的方法， 在整个生物科学中的功能遗传学研究的步伐。目前的做法侧重于 开发核酸酶，如TALEN、锌指和Cas蛋白，但这些的效用有限。 酶，它们都依赖于宿主内源性DNA修复机制。此外，使用核酸酶来 实现治疗性基因编辑需要额外的努力来防止脱靶诱变事件。一 针对这一挑战的部分解决方案已经以移动的遗传元件（MGE）的形式存在，这些系统 微生物已经使用（或斗争）了几千年，以实现精确的基因组操纵。我们将探讨 我们可以使用不同的MGE池，并鉴定具有可用于真核基因组编辑的特征的那些。 精确的基因组操作可以大致分为三个亚类：插入、缺失和插入。 重排，其中每一个都构成了一系列应用程序的基础。我们会考虑 这些操作模式中的每一个单独地首先选择候选MGE或这些系统的组件 通过生物信息学分析和从已知系统推断。例如，转座酶和 重组酶可以用于插入，而RNA模板化的基因组解扰发生在重组酶中。 纤毛虫可能适合于适应基因组重排。一旦这三种模式的候选人 我们将在体外对其进行功能表征，然后开发它们用于基因组编辑。 最后，我们将评估这些新型精确基因组编辑器在体内的特异性、有效性和安全性。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、独立的酶工具来克服对核酸酶的依赖， 基因组操作的操作模式。这项工作将补充我们正在进行的研究工作， 基因组工程组分的有效递送方法。这些技术将使我们能够 实现基因组工程的全部潜力，特别是作为治疗人类疾病的途径。