Plasmid hypermutation using a targeted artificial DNA replisome.
Xiao YiJoleen KheyRomas J KazlauskasMichael TravisanoPublished in: Science advances (2021)
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 a targeted artificial DNA replisome (TADR) that operates in live cells to processively replicate one strand of a plasmid with errors. It enhanced mutation rates of the 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 developing biologic drugs and industrial catalysts.
Keyphrases
- crispr cas
- escherichia coli
- amino acid
- single molecule
- protein protein
- circulating tumor
- rheumatoid arthritis
- induced apoptosis
- cell free
- cancer therapy
- mental health
- small molecule
- emergency department
- oxidative stress
- patient safety
- highly efficient
- drug delivery
- cell proliferation
- signaling pathway
- cell death
- nucleic acid
- human health
- electronic health record
- drug induced