Independent replication systems for rapid continuous evolution
We have engineered an orthogonal DNA replication (OrthoRep) system in yeast so that core properties of DNA replication can be freely manipulated in vivo without interfering with genomic replication. We are using OrthoRep for rapid targeted mutation and recombination of any desired gene(s) entirely in vivo. Recently, we have increased OrthoRep’s mutation rate so that it is ~100,000-times more mutagenic than the genome. We have used OrthoRep as a scalable directed evolution technology for hundreds of parallel rapid evolution experiments. We have applied OrthoRep to enzyme evolution, to studying the “solution space” for drug resistance, and to evolve antibodies. In addition, we are using OrthoRep as the foundation for a non-DNA episome, which should have applications in biocontainment as well as the discovery of new functional polymers.
Recording non-genetic information into DNA with rapid mutation systems
We have invented a genetic system called CHYRON (Cell HistorY Recording by Ordered iNsertion) that progressively accumulates short insertions of random nucleotides (nts) at a synthetic locus in the DNA of mammalian cells. Since daughter cells inherit the synthetic locus of their parents and then add additional nts to distinguish themselves, single-cell resolution lineage relationships can in principle be deduced from the mutational relationships among sequences retrieved through NGS. We have already shown, in cell culture, that one can use our synthetic locus to accurately reconstruct lineage relationships among groups of cells subject to complex splitting processes as well as to record the exposure of cells to arbitrary signals linked to insertional mutagenesis at the barcode. We are in the process of creating genetically engineered mice containing the barcode locus to study organ development and are also using our barcode in cancer models to study how tumors develop and metastasize.
Directed evolution using synthetically expanded genetic codes
Nature’s genetic code specifies 20 amino acids for all protein synthesis. However, recent efforts have achieved organisms with synthetically expanded genetic codes that specify one or more unnatural amino acids in addition to the 20 canonical amino acids. If we let evolution use these expanded genetic systems, will new (perhaps radically new) biological function emerge? We explore this possibility by using cells with expanded genetic codes to engineer and evolve novel functional proteins, including highly-sulfated antibodies and, most recently, peptides that trigger plant immunity. This effort will not only allow us to generate useful biomolecules, it also explores the idea that synthetic genetic codes with new chemistries can be superior to nature’s code.
Entirely unnatural peptide and polymer synthesis by expanded genetic codes
The holy grail in the expanded genetic code field is to achieve the ribosomal synthesis of entirely unnatural complex polymers. We are exploring the possibility of running more than one genetic code in the cell to achieve this, focusing on engineering the mitochondrion. In the process, we also seek greater understanding of mitochondria.
Studying and exploiting the biochemistry of an unusual DNA replication system
We are interested in understanding the genetics, biochemistry, and mechanistic aspects of an autonomous DNA replication system, called the pGKL1/2 (p1/p2) plasmids. This replication system serves as the basis of OrthoRep, which is the primary reason why it is interesting to us. However, the p1/p2 system also has 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 may also be useful for applications in biotechnology and gene therapy.