Improved bacterial recombineering by parallelized protein discovery
Timothy M. Wannier, Ákos Nyerges, Helene Kuchwara, Márton Simon Czikkely, D. Balogh, Gabriel Filsinger, Nathaniel C. Borders, Christopher Gregg, Marc J. Lajoie, Xavier Rios, Csaba Pál, George M. Church
Abstract
Significance Bacterial recombineering allows researchers to interrogate microbes by modifying their genomic DNA. Improvements to the efficiency of recombineering have allowed many simultaneous edits to be made at once. Here we describe "serial enrichment for efficient recombineering" (SEER), a method for identifying efficient single-stranded DNA-annealing proteins (SSAPs) in a microbe of interest. We use SEER to identify two SSAPs: 1) CspRecT doubles editing efficiency over Redβ, the state-of-the-art in Escherichia coli recombineering; and 2) PapRecT achieves high efficiency in Pseudomonas aeruginosa , a widely studied human pathogen. We show that these SSAPs work effectively across a broad range of Gammaproteobacteria, demonstrate vastly improved performance in multiplex applications, and provide broad host-range plasmid resources.