Evolutionary Innovation By Hypermutation Using a Targeted Artificial DNA Replisome

Extensive exploration of a protein's sequence space for improved or new molecular functions requires in vivo evolution with large populations. But disentangling the evolution of a target protein from the rest of the proteome is challenging. Here we designed a protein complex of Targeted Artificial DNA Replisome (TADR) in live cells to processively replicate one strand of a plasmid with errors. It enhanced mutation rates of target plasmid up to 2.3×10⁵-fold with only a 78-fold increase in off-target mutagenesis. It was used to evolve itself to increase error rate and increase the efficiency of an efflux pump while simultaneously expanding the substrate repertoire. TADR enables multiple simultaneous substitutions to discover functions inaccessible by accumulating single substitutions, affording potential for solving hard problems in molecular evolution and development of biologic drugs and industrial catalysts.