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Conservative Site-specific Recombination and Phase Variation

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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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Related Experiment Video

Updated: Apr 18, 2026

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Published on: May 15, 2018

Diversity-generating retroelements for programmable targeted hypermutagenesis.

Paul Rochette^1,2, Elena Lopez-Rodriguez^1,3, David J Wen^4,5

¹Institut Pasteur, Synthetic Biology Unit, Université Paris Cité, Paris, France.

Nature Biotechnology

|April 16, 2026

Summary

This summary is machine-generated.

Diversity-generating retroelements (DGRs) coupled with recombineering (DGRec) enable targeted DNA diversification in E. coli. This method accelerates the evolution of bacterial functions and can be adapted for yeast.

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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Related Experiment Videos

Last Updated: Apr 18, 2026

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

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Published on: May 15, 2018

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

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Published on: January 8, 2015

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Area of Science:

Molecular Biology
Synthetic Biology
Microbial Evolution

Background:

Diversity-generating retroelements (DGRs) are natural systems that drive rapid evolution of bacterial functions.
Targeted hypermutation is key to exploring sequence space for functional adaptation.

Purpose of the Study:

To develop a versatile method for sequence diversification using DGRs and recombineering.
To characterize DGR reverse transcriptase biases for efficient and controlled mutagenesis.
To demonstrate the application of this method in engineering biological systems.

Main Methods:

Coupling DGRs with recombineering (DGRec) in Escherichia coli.
Characterizing reverse transcriptase sequence biases and error rates.
Applying DGRec to engineer phage λ, dCas9 variants, and nanobodies.
Adapting DGR-mediated mutagenesis for yeast.

Main Results:

DGRec enables efficient diversification of user-defined DNA sequences (50-200 bp) in E. coli.
High mutation rates (up to 1.38 × 10^-2 per base per generation) were achieved, allowing multiple mutations.
Successful applications include phage host-range engineering and dCas9/nanobody evolution.
Feasibility of DGR-mediated mutagenesis was established in yeast.

Conclusions:

DGRec is a powerful tool for accelerating protein and functional element evolution.
The method offers precise control over mutation rates and locations.
DGRec expands the toolkit for synthetic biology and protein engineering across different organisms.