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.

项目摘要 连续定向进化（CDE）方法的最新发展使得越来越有可能 产生具有根本改变的甚至新的功能的生物分子，能够解决未满足的需求， 医学、生物技术和合成生物学。通过改造传统的逐步过程的经典 直接进化成一个在细胞中持续运作的过程，这些CDE方法，如噬菌体辅助 持续进化（PACE）理论上可以在进化过程中实现广泛的速度，规模和深度。 搜索然而，实施PACE（和其他CDE技术）的技术限制限制了 仅仅通过这些方法实际上可以实现什么。为了克服这些限制，我们的合作伙伴 团队最近建立了ePACE，这是一项新技术，将PACE与自动化、可扩展和 可定制的连续培养平台，称为eVOLVER。通过加入基础设施和流体 为了满足PACE对eVOLVER的要求，我们克服了传统PACE的许多限制， 生物分子的自动化、平行和连续进化的途径。使用ePACE，我们已经 成功地产生了生物医学相关的分子，包括Cas9的多重进化， 在以前无法访问的基因组靶位点进行精确的基因编辑。在这个项目中，我们将推进 ePACE在两个关键方面的能力-规模和可访问性-通过新的硬件和流体 技术发展。这些发展将使新的CDE计划能够应对新的 生物分子工程的挑战。我们将应对的第一个挑战是针对目标的工程系统 在哺乳动物细胞中整合大的基因大小的DNA有效载荷，这将使各种生物医学 本发明的方法可用于各种应用，包括用于几乎任何功能丧失性疾病的治疗性治疗。我们将申请ePACE， CRISPR相关转座酶（CAST）的高度并行化进化-最近发现，多个- 组件系统，使大DNA在细菌中的可编程整合-产生变异， 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的发展， 前所未有的规模，并大大增加了探索的进化轨迹的数量。第二 挑战是建立可推广的方法来进化蛋白质，以紧密和选择性地结合小分子， 分子配体，这将使不同的生物医学应用，包括生物传感和检测， 代谢工程，药物和毒素隔离。作为这一目标的一部分，我们将实现更广泛的 通过开发小型化、超低成本的eVOLVER变体，实现CDE民主化的使命， PACE功能，并使用它来建立一般管道，这些管道可以很容易地被实验室采用， 财务和技术费用。总之，这项工作将大大扩展CDE的能力， 为未满足的生物医学需求生产定制的生物分子。