Optimisation strategies for directed evolution without sequencing

Abstract

Directed evolution can enable engineering of biological systems with minimal knowledge of their underlying sequence-to-function relationships. A typical directed evolution process consists of iterative rounds of mutagenesis and selection that are designed to steer changes in a biological system (e.g. a protein) towards some functional goal. Much work has been done, particularly leveraging advancements in machine learning, to optimise the process of directed evolution. Many of these methods, however, require DNA sequencing and synthesis, making them resource-intensive and incompatible with developments in targeted in vivo mutagenesis. Operating within the experimental constraints of established sorting-based directed evolution techniques (e.g. Fluorescence-Activated Cell Sorting, FACS), we explore approaches for optimisation of directed evolution that do not require sequencing information. We then expand our methods to the context of emerging experimental techniques in directed evolution, which allow for single-cell selection based on fitness objectives defined from any combination measurable traits. Finally, we validate the developed selection strategies on the GB1 empirical landscape, demonstrating that they can lead to up to a 7.5 fold increase in the probability of attaining the global fitness peak. Author summaryThe standard approach to sorting-based selection in directed evolution is to take forward only the top-performing variants from each generation of a single population. In this work, we begin to explore alternative selection strategies within a simulated directed evolution framework. We propose "selection functions", which allow us to tune the balance of exploration and exploitation of a fitness landscape, and we demonstrate that splitting a population into sub-populations can improve both the likelihood and magnitude of a successful outcome. We also propose strategies to leverage emerging selection methods that can implement single-cell selection based on any combination of measurable traits. We validate our optimised directed evolution approaches on the empirical fitness landscape of the GB1 immunoglobulin protein.