We have engineered an orthogonal DNA replication (OrthoRep) system in yeast consisting of an error-prone DNA polymerase that replicates a special DNA plasmid without increasing the mutation rate of the genome. This architecture liberates the replication of chosen genes from the low mutational speed limits of genome propagation. Chosen genes encoded on the special DNA plasmid autonomously hypermutate and evolve in vivo, allowing us to accelerate macromolecular evolution by thousands to millions fold at scale. With OrthoRep, we can systematically and prospectively watch, perturb, and apply evolution on laboratory timescales. We have used OrthoRep to evolve enzymes, biosensors, and antibodies, and to study the “fitness landscapes” on which macromolecular sequences travel. We focus on three key basic science questions: What does the map between macromolecular sequence and function look like? How are essentially-infinite high dimensional sequence spaces (such as those defining RNA and protein function) productively searched? How does a gene’s evolutionary past shape its future? We focus on two key application areas: sustainability through the evolution of new biocatalysts; human health through the evolution of new macromolecular therapeutics.
Recording transient non-genetic information into DNA
We have invented genetic systems called CHYRON (Cell HistorY Recording by Ordered iNsertion) and peCHYRON (prime editing CHYRON) that progressively accumulate short insertions of random nucleotides (nts) at a synthetic locus in the DNA of mammalian cells in an ordered manner. Since daughter cells inherit the CHYRON locus of their parents and then add additional nts to distinguish themselves, deep lineage relationships can be deduced by sequencing CHYRON loci. Since the addition of nts can be linked to cellular stimuli, CHYRON can log the history of events a cell experiences, to be later read by sequencing. We are using CHYRON to study normal and cancer development in animals, focusing on the key question: How does a cell’s past influence its developmental future?
Directed evolution of artificial proteins and polymers
We are interested in studying how artificially designed proteins that are divorced from the natural evolutionary history of extant proteins evolve. We are also interested in understanding the behavior and evolvability of novel proteins containing building blocks that nature has not used before, achieved through expanded genetic codes.We focus on three key questions: Are the statistical features defining sequence-function relationships for artificial proteins meaningfully different from those defining natural proteins? Are artificial proteins less or more evolvable than natural proteins and if so, why? Can artificial proteins access radically new functions?
Studying atypical DNA replication systems
We are interested in understanding the genetics, biochemistry, and mechanistic aspects of an autonomous protein-primed DNA replication system, the pGKL1/2 (p1/p2) plasmids. This replication system serves as the basis of OrthoRep, which is the primary reason it is interesting to us. Additionally, the p1/p2 system also has a number of unusual biochemical and molecular properties that make it unique among DNA replication systems. Studying the basic biology of this plasmid system may give us fundamental insights on the mechanisms of DNA replication, repair, and segregation. Some of p1/p2’s unusual properties, in particular the covalent link of DNA to proteins, may also be useful for applications in biotechnology and gene therapy.