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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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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

Directed evolution of recombinase specificity by split gene reassembly.

Charles A Gersbach¹, Thomas Gaj, Russell M Gordley

¹The Skaggs Institute for Chemical Biology, Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Nucleic Acids Research

|March 3, 2010

Summary

Researchers engineered novel enzymes called site-specific recombinases for precise DNA modification. This breakthrough advances gene therapy and genetic research by enabling efficient genome editing with high sensitivity and specificity.

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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

Area of Science:

Molecular Biology
Enzyme Engineering
Genetics

Background:

Efficient and specific DNA modification is crucial for advancing gene therapies and genetic studies.
Developing novel enzymes with tailored substrate specificities is a key challenge in molecular biology.

Purpose of the Study:

To develop a robust strategy for evolving site-specific recombinases with novel substrate specificities.
To engineer enzymes capable of precise DNA sequence modification for research and clinical applications.

Main Methods:

A stringent evolution system was employed, using enzyme-mediated reassembly of the beta-lactamase gene for selection.
Recombinase variants were selected for activity on new substrates, altering catalytic domains within a modular zinc finger-recombinase fusion protein.
The system demonstrated tunable selectivity and high sensitivity, with gene reassembly detectable over several orders of magnitude.

Main Results:

Engineered recombinases were successfully evolved to target specific sequences within the human genome in just three rounds of selection.
The evolution method identified conserved residues among recombinase family members, indicating a robust engineering strategy.
The developed system achieved exceptional sensitivity and tunable selectivity for DNA modification.

Conclusions:

The enhanced evolution system provides a practical and expedient method for recombinase engineering and genome editing.
This approach has significant potential for diverse applications in academic, industrial, and clinical settings.
The engineered recombinases offer precise DNA modification capabilities, paving the way for improved gene therapies and genetic studies.