Evolving Strand-Displacing Polymerases with Isothermal CSR

Compartmentalized self-replication (CSR) has proven useful for DNA polymerase evolution by creating a feedback loop in which a polymerase replicates its own DNA in an isolated emulsified compartment.  This method has been used to evolve a number of enhanced phenotypes, including increased thermostability, inhibitor tolerance, and unnatural and modified base incorporation. However, CSR currently relies on thermal cycling and PCR. We hope to evolve polymerases that can function better in the context of isothermal amplification reactions, including polymerases with improved strand displacement activity. We therefore modified CSR for an isothermal amplification reaction, rolling circle amplification (RCA), in which the DNA polymerase must continuously displace downstream base-paired DNA in order to effectively replicate its own gene.  Starting from a shuffled library of the DNA polymerases from Thermus aquaticus (Taq) and Bacillus stearothermophilus (Bst), 5 rounds of selection have yielded functional chimeric variants (65%), in which the polymerase best known for isothermal amplification, Bst, has acquired small Taq insertions. We observed Taq insertions at residues 530 to 535 in 71% of the population and at residues 494 to 497 in 29% of the population, indicating that positive selection is occurring. Some 16% of the sequenced population contained additional mutations known to increase thermostability or inhibitor resistance. Compared to Bst, the successful chimeras showed increased amplification rates, improved strand displacement activity, and greater thermostability in isothermal amplification assays. Surprisingly, they also showed increased activity in PCR reactions with difficult templates.  These experiments represent the first directed evolution of a polymerase at a single temperature, opening up new avenues for improving amplification-based point-of-care diagnostics.