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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Cells are sometimes infected by more than one virus at once. When two viruses disassemble to expose their genomes for replication in the same cell, similar regions of their genomes can pair together and exchange sequences in a process called recombination. Alternatively, viruses with segmented genomes can swap segments in a process called reassortment.
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Related Experiment Video

Updated: Oct 29, 2025

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker
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RecT Recombinase Expression Enables Efficient Gene Editing in Enterococcus spp.

Victor Chen1, Matthew E Griffin1, Pascal Maguin2

  • 1Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller Universitygrid.134907.8, New York, New York, USA.

Applied and Environmental Microbiology
|July 7, 2021
PubMed
Summary
This summary is machine-generated.

RecT recombinase enhances gene editing in Enterococcus faecium, enabling faster and more efficient genetic studies of this important bacterium. This breakthrough aids research into both beneficial and pathogenic strains.

Keywords:
CRISPRE. faeciumEnterococcusVREbiotechnologygeneticsrecombineering

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Area of Science:

  • Microbiology
  • Genetics
  • Molecular Biology

Background:

  • Enterococcus faecium is a versatile bacterium with both beneficial commensal roles and pathogenic, antibiotic-resistant strains.
  • Current gene-editing methods for E. faecium are inefficient, hindering functional and mechanistic studies.
  • Understanding the genetic basis of E. faecium's diverse activities is crucial due to its public health significance.

Purpose of the Study:

  • To develop improved gene-editing technologies for Enterococcus species.
  • To enhance the efficiency of recombineering in both commensal and antibiotic-resistant E. faecium strains.
  • To facilitate genetic studies for characterizing E. faecium functions and mechanisms.

Main Methods:

  • Expression of Enterococcus faecium RecT recombinase to improve recombineering efficiency.
  • Combined use of RecT, CRISPR-Cas9, and guide RNAs (gRNAs) for scarless single-stranded DNA recombineering.
  • Utilized RecT for chromosomal insertions of DNA templates for gene deletion mutants.
  • Employed CRISPR-Cas9-mediated recombineering to knock out sortase A genes.

Main Results:

  • RecT expression significantly boosted recombineering efficiency in various Enterococcus species.
  • CRISPR-Cas9-RecT system enabled highly efficient scarless gene editing in E. faecium.
  • RecT facilitated efficient chromosomal DNA insertions for generating gene deletion mutants.
  • Successful knockout of sortase A genes demonstrated the utility of the developed methods.

Conclusions:

  • RecT-mediated recombineering provides a rapid and efficient method for genetic manipulation in E. faecium.
  • These improved methods will accelerate functional and mechanistic studies of Enterococcus species.
  • The developed techniques address limitations in current genetic engineering approaches for E. faecium.