PT  - JOURNAL ARTICLE
AU  - Timothy M. Wannier
AU  - Akos Nyerges
AU  - Helene M. Kuchwara
AU  - Márton Czikkely
AU  - Dávid Balogh
AU  - Gabriel T. Filsinger
AU  - Nathaniel C. Borders
AU  - Christopher J. Gregg
AU  - Marc J. Lajoie
AU  - Xavier Rios
AU  - Csaba Pál
AU  - George M. Church
TI  - Improved bacterial recombineering by parallelized protein discovery
AID  - 10.1101/2020.01.14.906594
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.01.14.906594
4099  - http://biorxiv.org/content/early/2020/01/16/2020.01.14.906594.short
4100  - http://biorxiv.org/content/early/2020/01/16/2020.01.14.906594.full
AB  - Exploiting bacteriophage-derived homologous recombination processes has enabled precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, Multiplex Automated Genome Engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, highly efficient SSAPs must first be found. Here we introduce a high-throughput method for SSAP discovery that we call Serial Enrichment for Efficient Recombineering (SEER). By performing SEER in E. coli to screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5 to 10-fold in E. coli, while PapRecT enables efficient recombineering in Pseudomonas aeruginosa, a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.