Project Summary/Abstract Over the past five years, my lab has made significant strides in the development of genetic systems capable of driving the rapid mutation and evolution of user-selected genes of interest in vivo. These systems have allowed us and others to quickly and scalably evolve enzymes, proteins, and antibodies to address a range of problems spanning from studying drug resistance to creating affinity reagents on demand. These systems have also begun to allow us to use rapid mutational accumulation as a method for tracing cell lineage in developing animals. One of our key accomplishments has been the invention of an orthogonal DNA replication system (OrthoRep). In OrthoRep, an error-prone orthogonal DNA polymerase (DNAP) exclusively replicates a special cytosolic plasmid encoding only genes of interest (GOIs), driving their continuous evolution fully in vivo. This MIRA will integrate our lab’s work on OrthoRep and support its further development and application in the next five years. In particular, we will grow the core OrthoRep technology in order to accelerate GOI evolution in yeast even more than we currently have, attempt to establish OrthoRep in mammalian cells in order to extend the range of problems OrthoRep can directly address, apply OrthoRep to the engineering of bespoke Cas9s to extend the range and efficacy of targeting, apply OrthoRep to improve the capabilities of a lineage tracing tool developed by my lab, and apply OrthoRep to the generation of mutually orthogonal collections of aminoacyl- tRNA synthetase (aaRS)/tRNA pairs to support genetic code expansion efforts also ongoing in my lab. We hope the set of activities proposed for this MIRA will solidify OrthoRep as an exceptionally powerful genetic system for evolving enzymes and proteins capable of solving high-reward problems in the chemical, biological, and biomedical sciences.