An Adaptable Platform for Directed Evolution in Human Cells

Abstract

The discovery and optimization of biomolecules that reliably function in metazoan cells is imperative for both the study of basic biology and the treatment of disease. We describe the development, characterization, and proof-of-concept application of a platform for directed evolution of diverse biomolecules of interest (BOIs) directly in human cells. The platform relies on a custom-designed adenovirus variant lacking multiple genes, including the essential DNA polymerase and protease genes, features that allow us to evolve BOIs encoded by genes as large as 7 kb while attaining the mutation rates and enforcing the selection pressure required for successful directed evolution. High mutagenesis rates are continuously attained by trans-complementation of a newly engineered, highly error-prone form of the adenoviral polymerase. Selection pressure that couples desired BOI functions to adenoviral propagation is achieved by linking the functionality of the encoded BOI to the production of adenoviral protease activity by the human cell. The dynamic range for directed evolution can be enhanced to several orders of magnitude via application of a small molecule-based adenoviral protease inhibitor to modulate selection pressure during directed evolution experiments. This platform makes it possible, in principle, to evolve any biomolecule activity that can be coupled to adenoviral protease expression or activation by simply serially passaging adenoviral populations carrying the BOI. As proof-of-concept, we use the platform to evolve, directly in the human cell environment, several transcription factor variants that maintain high levels of function while gaining resistance to a small molecule inhibitor. We anticipate that this platform will substantially expand the repertoire of biomolecules that can be reliably and robustly engineered for both research and therapeutic applications in metazoan systems.

A high-throughput platform for feedback-controlled directed evolution

“…Though the current method is presently consumable-intensive when operating at maximum capacity (96 lagoons), further method development could greatly extend the hands-off user window above our standard operating time frames of 7, 14 or 21 hours by implementing tip cleaning cycles. While currently designed primarily for phage-assisted continuous evolution, it should also be feasible to extend the system to continuous diversification platforms such as those used with yeast 2,22 and mammalian cells 3,4 when utilized within a sterilized, laminar flow environment. In the future, PRANCE could be implemented to perform previously infeasible evolution experiments such as the characterization of complex fitness landscapes, the evolution of small molecule-dependent proteins, and experiments integrating responsive negative selections-experiments facilitated by the PRANCE-specific features of multiplexing, volume miniaturization, and feedback control, respectively.…”

“…Continuous directed evolution enables rapid population diversification and selection by coupling a single desired protein function to fitness, and has been used to engineer biological systems that are not tractable with rational design alone. [1][2][3][4][5] The most well-established continuous evolution method, Phage-Assisted Continuous Evolution (PACE), 6 has been successfully applied to a diverse set of protein engineering challenges, including the evolutions of proteases with altered substrate specificity, 7 of proteins with improved soluble expression, 8 and of CRISPR-Cas proteins with broadened PAM recognition. 9,10 The power of this technique comes from the ability to perform many rounds of directed evolution in a single day, making it ideal for challenging evolution goals that require numerous sequential mutations.…”

Combining rational design and continuous evolution on minimalist proteins that target DNA

“…Other systems for continuous evolution are being developed that should allow researchers opportunities and flexibility for incorporating evolution methods, including those for continuous evolution in human and mammalian cell systems. 67,68 Such systems may facilitate development of proteins useful in human medical interventions, as well as veterinary care. As Max and E47 are found in humans and mammals, continuous evolution in these advanced eukaryotic systems may reveal mutations that allow MEF to perform better in mammalian/human cells, and can allow close examination of MEF's protein-partner preferences under selective pressure in the presence of competing endogenous bHLH, bHLHZ, and bZIP proteins.…”

Genetic Engineering by DNA Recombineering

“…This article aims to distill the many possible iterations down to just three simple protocols that-based on our extensive experience engineering adenovirus vectors (Berman et al, 2018;Wong et al, 2018), E. coli genomes , and other DNA sequences-function reliably and efficiently. In particular, the multitude of available recombineering-based methods and their many variations can make it daunting for a new user to choose the best option for a specific purpose.…”

“…Typical applications of recombineering include modifying the E. coli genome itself or the genomes of large human or animal viruses as BACs in E. coli, which are later reintroduced into the host organism (Berman et al, 2018;Moore, Papa, & Shoulders, 2018;Narayanan & Chen, 2011). The modified genomes can then produce genetically engineered virions in a process known as "viral rescue."…”