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.
