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.

PROJECT NARRATIVE We have developed methods to evolve proteins continuously in the laboratory toward new biological activities. These methods offer tremendous potential for evolving next-generation gene editing tools and protein therapeutics to treat human diseases; however, they are limited by technical barriers that negatively impact their success rate, efficiency, and broader application. To address these limitations, we propose to develop powerful automation technologies that will dramatically increase the speed, scale, and wider adoption of these methods, and to use the resulting technologies to evolve two classes of proteins with therapeutic potential.