Continuous evolution of user-defined genes at 1 million times the genomic mutation rate.

When nature evolves a gene over eons at scale, it produces a diversity of homologous sequences with patterns of conservation and change that contain rich structural, functional, and historical information about the gene. However, natural gene diversity accumulates slowly and likely excludes large regions of functional sequence space, limiting the information that is encoded and extractable. We introduce upgraded orthogonal DNA replication (OrthoRep) systems that radically accelerate the evolution of chosen genes under selection in yeast.

Directed evolution of aminoacyl-tRNA synthetases through hypermutation.

Genetic code expansion (GCE) has become a critical tool in biology by enabling the site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Central to GCE is the development of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs wherein engineered aaRSs recognize chosen ncAAs and charge them onto tRNAs that decode blank codons ( ., the amber stop codon).

Continuous evolution of user-defined genes at 1-million-times the genomic mutation rate.

When nature maintains or evolves a gene's function over millions of years at scale, it produces a diversity of homologous sequences whose patterns of conservation and change contain rich structural, functional, and historical information about the gene. However, natural gene diversity likely excludes vast regions of functional sequence space and includes phylogenetic and evolutionary eccentricities, limiting what information we can extract. We introduce an accessible experimental approach for compressing long-term gene evolution to laboratory timescales, allowing for the direct observation of extensive adaptation and divergence followed by inference of structural, functional, and environmental constraints for any selectable gene.

Systems for in vivo hypermutation: a quest for scale and depth in directed evolution.

Traditional approaches to the directed evolution of genes of interest (GOIs) place constraints on the scale of experimentation and depth of evolutionary search reasonably achieved. Engineered genetic systems that dramatically elevate the mutation of target GOIs in vivo relieve these constraints by enabling continuous evolution, affording new strategies in the exploration of sequence space and fitness landscapes for GOIs. We describe various in vivo hypermutation systems for continuous evolution, discuss how different architectures for in vivo hypermutation facilitate evolutionary search scale and depth in their application to problems in protein evolution and engineering, and outline future opportunities for the field.

Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities.

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales.