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Efficient Genome Manipulation by Variants of Site-Specific Recombinases R and TD
Eugenia Voziyanova1, Rachelle P Anderson1, Riddhi Shah1
1School of Biosciences, Louisiana Tech University, 1 Adams Boulevard, Ruston, LA 71272, USA.
Journal of Molecular Biology
|November 12, 2015
Summary
Researchers engineered new variants of yeast tyrosine recombinases, R and TD, for enhanced genome engineering in bacteria and mammalian cells. These modified enzymes show improved activity and enable complex genetic manipulations at multiple sites.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Genome engineering relies on site-specific DNA modifying enzymes for complex applications.
- While eight yeast tyrosine recombinases exist, only Flp recombinase is widely used.
- Expanding the repertoire of recombinases is crucial for advancing genome engineering.
Purpose of the Study:
- To engineer and analyze variants of yeast tyrosine recombinases R and TD.
- To assess their suitability for genome engineering in diverse cell types, including mammalian cells.
- To investigate modifications enhancing enzyme performance and expanding their utility.
Main Methods:
- Engineering variants of yeast tyrosine recombinases R and TD.
- Testing enzyme activity in bacterial (Escherichia coli) and mammalian cell systems.
- Evaluating performance in various recombination reactions: excision, integration, and cassette exchange.
Main Results:
- Engineered R and TD variants exhibit activity suitable for genome engineering in E. coli and mammalian cells.
- Shortening the C-terminus of R recombinase unexpectedly improved its performance.
- Engineered variants demonstrated activity in excision, integration, and dual recombinase-mediated cassette exchange.
Conclusions:
- Engineered R and TD recombinase variants significantly expand the toolkit for genome engineering.
- These variants offer improved functionality in bacterial and mammalian systems.
- Further understanding of recombinase structure-activity relationships can optimize genome editing tools.
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