Development of the eVOLVER continuous culture platform for high-throughput phage-assisted continuous evolution
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Abstract
Continuous directed evolution (CDE) has emerged as a powerful paradigm for generating biomolecules toward radically altered or even new functions capable of addressing unmet needs in medicine, biotechnology, and synthetic biology. Indeed, 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 PACE alone. Here, I combine the continuous hypermutation and selection advantages of PACE with the automated, scalable, and customizable eVOLVER continuous culture platform to create ePACE. ePACE overcomes many of the design and operational challenges of traditional PACE to facilitate parallel, automated, and continuous evolution of biomolecules directly on the benchtop. ePACE is applied to the evolution of Nme2Cas9 to target single-nucleotide-pyrimidine PAM sequences via high-throughput parallel evolution against individual PAM sequences, resulting in four highly-active Cas9 variants.