Continuous Directed Evolution in Mammalian Cells

Abstract

Directed evolution is a powerful methodology for the creation of new biomolecules with user-desired functions. Most directed evolution experiments are performed in vitro, in bacteria, or in yeast, even when the evolved biomolecule is intended to function in mammalian cells. As a result, the functions of biomolecules evolved in these environments are often derailed in the complex mammalian cellular environment. The development of highly efficacious methods for directed evolution in mammalian cells has severely lagged behind similar methods in single-celled organisms, owing to the relative difficulties of both mammalian cell culture and genomic engineering in mammalian cells. In this thesis, I describe the development and subsequent application of a high-throughput, adaptable, virus-based continuous directed evolution method that uses the mammalian cell for simultaneously mutagenizing, expressing, and selecting an evolving gene of interest. This platform functions by making adenoviral propagation in mammalian cells dependent upon the activity of a virally encoded gene of interest, which is continuously mutagenized by a highly error-prone engineered adenoviral polymerase. We demonstrated the platform’s efficacy in proof-of-principle evolution experiments by evolving a transcription factor to be insensitive to a small molecule inhibitor. We then engineered selection circuits for evolving endogenous human G-protein coupled receptors, wherein viral replication is coupled to an endogenous signaling pathway. We also engineered selection circuits for evolving exogenous CRISPR systems, wherein viral replication is coupled to an exogenous transcriptional couple. For both selection circuits, we demonstrated selection pressure sufficient to drive a directed evolution campaign through viral replication assays. Finally, we highlight a wide range of biomolecules for which directed evolution mammalian cells would be impactful, but was previously out of reach before the development of virus-based continuous evolution methods.