ePACE: automation platforms for adaptable and scalable continuous evolution of biomolecules with therapeutic potential

ePACE：自动化平台，用于具有治疗潜力的生物分子的适应性和可扩展的持续进化

• 批准号: 10734591
• 负责人: Ahmad Samir Khalil
• 金额: $ 87.15万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-03 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10734591
• 关键词: Address; Adopted; Adoption; Affinity; Automation; Bacteria; Bacteriophages; Binding; Binding Proteins; Biological; Biosensing Techniques; Biotechnology; Cells; Chemicals; Clinical Treatment; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Integration; Detection; Development; Dimensions; Directed Molecular Evolution; Disease; Engineering; Evolution; Factor IX; Generations; Genes; Genetic Diseases; Genome; Genomics; Genotype; Goals; Guide RNA; Head; Hemophilia B; Hepatocyte; Homologous Gene; Human; Human Cell Line; Human Genome; Laboratories; Ligands; Mammalian Cell; Medicine; Methods; Mission; Mutation; Pathway interactions; Pharmaceutical Preparations; Population; Process; Protein Engineering; Proteins; Protocols documentation; Reporting; Robotics; Schedule; Scheme; Site; Speed; Standardization; System; Techniques; Technology; Therapeutic; Toxin; Transgenes; Transposase; Variant; Work; antibody engineering; arm; cancer immunotherapy; cellular engineering; combinatorial; cost; design; genomic locus; human disease; improved; interest; loss of function; mammalian genome; metabolic engineering; miniaturize; new technology; next generation; novel; operation; parallelization; small molecule; success; synthetic biology; technology development; theories; therapeutic genome editing; therapeutic protein; tool; virtual

PROJECT SUMMARY The recent development of continuous directed evolution (CDE) methods has made it increasingly possible to generate biomolecules with radically altered or even new functions capable of addressing unmet needs in medicine, biotechnology, and synthetic biology. By transforming the traditional stepwise process of classical directed evolution into one that operates continuously in cells, these CDE methods, such as Phage-Assisted Continuous Evolution (PACE), can theoretically enable extensive speed, scale, and depth in an evolutionary search. However, the technical limitations of implementing PACE (and other CDE techniques) have restricted what can be practically achieved with these approaches alone. To overcome these limitations, our collaborative team recently established ePACE, a new technology that combines PACE with an automated, scalable, and customizable continuous culture platform, called eVOLVER. By on-boarding the infrastructural and fluidic requirements of PACE onto eVOLVER, we overcame many of the limitations of traditional PACE and unlocked pathways for automated, parallel, and continuous evolution of biomolecules. Using ePACE, we already succeeded in generating biomedically-relevant molecules, including the multiplexed evolution of Cas9 for precision gene editing at previously-inaccessible genomic target sites. In this project, we will advance the capabilities of ePACE in two critical dimensions – scale and accessibility – through new hardware and fluidic technology developments. These developments will enable novel CDE schemes necessary to tackle new challenges in biomolecular engineering. The first challenge we will tackle is engineering systems for targeted integration of large, gene-sized DNA payloads in mammalian cells, which would enable diverse biomedical applications, including therapeutic treatments for virtually any loss-of-function disease. We will apply ePACE for highly parallelized evolution of CRISPR-associated transposases (CASTs) — recently discovered, multi- component systems that enable programmable integration of large DNA in bacteria — to generate variants with robust mammalian genomic integration activity. To effectively explore the combinatorial space of CAST components, we will develop an ultra-high-throughput eVOLVER variant that facilitates ePACE evolutions at unprecedented scale and dramatically increases the number of evolutionary trajectories explored. The second challenge is establishing generalizable methods to evolve proteins for tight and selective binding of small- molecule ligands, which would enable diverse biomedical applications, including biosensing and detection, metabolic engineering, and drug and toxin sequestration. As part of this goal, we will deliver on the broader mission of democratizing CDE by developing a miniaturized, ultra-low-cost eVOLVER variant that facilitates PACE functionality, and use it to establish general pipelines that can be easily adopted by labs with minimal financial and technical overhead. Together, this work will substantially expand the capabilities of CDE while producing bespoke biomolecules for unmet biomedical needs.

项目概要 连续定向进化（CDE）方法的最新发展使得越来越有可能 产生具有根本性甚至新功能的生物分子，能够满足未满足的需求 医学、生物技术和合成生物学通过改变传统的经典逐步过程。 这些 CDE 方法，例如噬菌体辅助方法，将进化定向为在细胞中连续运行的方法 持续进化（PACE），理论上可以在进化中实现广泛的速度、规模和深度 然而，实施 PACE（和其他 CDE 技术）的技术限制受到了限制。 为了克服这些限制，我们的合作可以实现什么。 团队最近建立了 ePACE，这是一项将 PACE 与自动化、可扩展和 可定制的连续培养平台，称为 eVOLVER。 将PACE的要求移植到eVOLVER上，我们克服了传统PACE的诸多限制并解锁了 使用 ePACE，我们已经实现了生物分子并行、连续进化的自动化途径。 成功生成生物医学相关分子，包括 Cas9 的多重进化 在这个项目中，我们将推进以前无法访问的基因组靶位点的精确基因编辑。 ePACE 通过新的硬件和流体在两个关键维度上的能力——规模和可访问性 这些发展将使新的 CDE 计划成为解决新问题所必需的。 我们要解决的第一个挑战是生物分子工程的工程系统。 将大型基因大小的 DNA 有效负载整合到哺乳动物细胞中，这将使多样化的生物医学成为可能 应用，包括几乎所有功能丧失疾病的治疗方法，我们将应用 ePACE 来治疗。 CRISPR 相关转座酶 (CAST) 的高度并行进化——最近发现的多 能够对细菌中的大DNA进行可编程集成的组件系统——以生成变体 强大的哺乳动物基因组整合活性，有效探索 CAST 的组合空间。 组件，我们将开发一种超高吞吐量的 eVOLVER 变体，以促进 ePACE 的进化 第二个是前所未有的规模，并显着增加了探索的进化轨迹的数量。 面临的挑战是建立通用方法来进化蛋白质，以紧密和选择性地结合小分子 分子配体，这将使多种生物医学应用成为可能，包括生物传感和检测， 作为这一目标的一部分，我们将实现更广泛的目标。 通过开发小型化、超低成本的 eVOLVER 变体来实现 CDE 民主化的使命 PACE 功能，并使用它来建立通用管道，实验室可以轻松采用 总之，这项工作将大大扩展 CDE 的能力，同时 生产定制生物分子以满足未满足的生物医学需求。