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Updated: Dec 20, 2025

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Improved bacterial recombineering by parallelized protein discovery.

Timothy M Wannier1, Akos Nyerges2,3, Helene M Kuchwara2

  • 1Department of Genetics, Harvard Medical School, Boston, MA 02115; timothy_wannier@hms.harvard.edu gchurch@genetics.med.harvard.edu.

Proceedings of the National Academy of Sciences of the United States of America
|May 30, 2020
PubMed
Summary

Researchers developed a new method, serial enrichment for efficient recombineering (SEER), to discover single-stranded DNA-annealing proteins (SSAPs). This enables precise genome engineering in more microbial species, expanding genetic variant construction.

Keywords:
MAGERecTgenome engineeringrecombineeringsynthetic biology

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Area of Science:

  • Microbiology
  • Synthetic Biology
  • Genomics

Background:

  • Bacteriophage recombineering enables precise, multiplex microbial genome editing.
  • Current methods like MAGE and DIvERGE are limited to specific hosts with known single-stranded DNA-annealing proteins (SSAPs).
  • Discovering efficient SSAPs is crucial for expanding genome engineering capabilities to new microbial hosts.

Purpose of the Study:

  • To develop a high-throughput method for discovering novel SSAPs.
  • To identify and characterize new SSAPs for enhanced microbial genome engineering.
  • To enable genome-scale engineering in previously intractable bacterial species.

Main Methods:

  • Developed and implemented a high-throughput screening method called serial enrichment for efficient recombineering (SEER).
  • Screened hundreds of putative SSAPs in *Escherichia coli* using the SEER method.
  • Validated the activity of identified SSAPs in *E. coli*, *Pseudomonas aeruginosa*, and other enterobacteria.

Main Results:

  • Identified two highly active SSAP variants: PapRecT and CspRecT.
  • CspRecT significantly increased single-locus editing efficiency (up to 50%) and multiplex editing (5- to 10-fold) in *E. coli*.
  • PapRecT enabled efficient recombineering in *Pseudomonas aeruginosa* and both SSAPs showed activity in other enterobacteria.

Conclusions:

  • The SEER method is effective for discovering novel and efficient SSAPs.
  • The identified SSAPs, CspRecT and PapRecT, significantly advance microbial genome engineering capabilities.
  • SEER deployment in new species will facilitate genotype-to-phenotype relationship studies in diverse bacteria.