Virus-assisted directed evolution of biomolecules

Abstract

Directed evolution is a powerful technique that uses principles of natural evolution to enable the development of biomolecules with novel functions. However, the slow pace of natural evolution does not support the demand for rapidly generating new biomolecular functions in the laboratory. Viruses offer a unique path to design fast laboratory evolution experiments, owing to their innate ability to evolve much more rapidly than most living organisms, facilitated by a smaller genome size that tolerate a high frequency of mutations, as well as a fast rate of replication. These attributes offer a great opportunity to evolve various biomolecules by linking their activity to the replication of a suitable virus. This review highlights the recent advances in the application of virus-assisted directed evolution of designer biomolecules in both prokaryotic and eukaryotic cells.

Keywords: Continuous evolution; Molecular evolution; PACE; PANCE; VADER; VEGAS.

Figure 1:

Phage-assisted continuous evolution (PACE). a, Overview of the PACE selection scheme. Selection phages (SP), with gIII replaced with the BOI gene, are used to infect E. coli cells containing accessory plasmid (AP) and mutagenesis plasmid (MP). The steady-state volume of the ‘lagoon’ is maintained through constant inflow of fresh host cells and outflow. The desired activity of the BOI is connected to the production of pIII: Active BOI mutants facilitate pIII expression, generating infectious progeny phage that continues to replicate, while inactive BOIs do not. b, Negative selection to deplete undesired phenotypes by linking their production to gIII-neg, expression of which leads to inactive progeny phage. c, Left: A one-hybrid system for evolving DNA-binding BOIs; Middle: A two-hybrid system for evolving protein binding partners; Right: A split-RNAP system for evolving/sensing protein-protein interactions. d, Left: A split-gIII, which is reconstituted post-translationally using trans-splicing inteins, allows dual positive selection. Right: Evolution of large base editor genes using the split-intein strategy

Figure 2:

Virus-assisted-directed-evolution in mammalian cells. a, Selection scheme for continuous evolution: Viruses with essential genes replaced by BOI gene are used to infect mammalian cells. Activity of BOI is linked to the expression of one of the deleted essential viral genes to enable selection of active variants. b, Early examples of HIV-assisted directed evolution. c, Continuous directed evolution using Adenovirus. d, Continuous directed evolution using Sindbis virus. e, AAV-assisted directed evolution of tRNAs that employ a novel double-sieve selection to isolate active-yet-orthogonal suppressor tRNA mutants for enhanced ncAA incorporation in mammalian cells.