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

项目总结 连续定向进化(CDE)方法的最新发展使其越来越有可能 产生具有根本改变甚至新功能的生物分子，能够解决未满足的需求 医学、生物技术和合成生物学。通过改造传统的循序渐进的过程 定向进化为在细胞中持续工作的方法，这些CDE方法，如噬菌体辅助 持续演进(PACE)，理论上可以在演进中实现广泛的速度、规模和深度 搜索。然而，实现PACE(和其他CDE技术)的技术限制已经受到限制 仅靠这些方法实际上能达到什么效果。为了克服这些限制，我们的协作 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 可定制的持续培养平台，称为Evolver。通过自注册基础设施和流体 在Evolver上对Pace的要求，我们克服了传统Pace的许多限制，解锁了 生物分子自动、并行和持续进化的途径。使用ePACE，我们已经 成功地产生了与生物医学相关的分子，包括Cas9对 在以前无法访问的基因组目标位置进行精确的基因编辑。在这个项目中，我们将推进 EPACE通过新硬件和流体在两个关键维度--规模和可访问性--方面的能力 技术发展。这些发展将使新的CDE计划成为必要，以应对新的 生物分子工程中的挑战。我们将解决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，这将使不同的生物医学 应用，包括对几乎任何功能丧失疾病的治疗。我们将申请ePACE CRISPR相关转座酶(CAST)的高度并行化进化--新近发现的多个 能够对细菌中的大DNA进行可编程整合的组件系统--以产生与 强大的哺乳动物基因组整合活动。有效地探索CAST的组合空间 组件，我们将开发超高吞吐量的Evolver变体，以促进ePACE的发展，网址为 前所未有的规模，极大地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，这将使包括生物传感和检测在内的各种生物医学应用成为可能， 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 通过开发一种小型化、超低成本的Evolver变体来促进CDE民主化的使命 Pace功能，并使用它建立实验室可以轻松采用的常规管道，只需最少的 财务和技术管理费用。总之，这项工作将极大地扩展CDE的能力，同时 为未得到满足的生物医学需求生产定制的生物分子。

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.