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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Tools for Improved Crop Biotechnology

Location: Crop Improvement and Genetics Research

Title: Gene stacking by recombinases

item Srivastava, Vibha
item Thomson, James - Jim

Submitted to: Plant Biotechnology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/24/2015
Publication Date: 7/28/2015
Citation: Srivastava, V., Thomson, J.G. 2015. Gene stacking by recombinases. Plant Biotechnology Journal. 14(2):471-482. doi: 10.1111/pbi.12459.

Interpretive Summary: Modification of plant genomes to improve crop productivity will provide food security for future generations. To facilitate the development of biotechnology, our labs have developed and tested a number of novel recombinases for genomic engineering in plants. These enzymes recognize specific DNA sequences and perform specific reactions to separate and rejoin DNA segments at those sites. We present evidence demonstrating that recombinase systems can mediate precise insertion of genes into chromosomes and deletion of unneeded DNA. We highlight the conservative nature of the recombinase enzymes in maintaining gene integrity and producing consistent gene expression in the progeny of genetically engineered plants. Our use of strategic designs allows sequential gene additions into the same site (“gene stacking”) and production of unique transgenic loci that can be readily incorporated by traditional breeding into cultivated varieties.

Technical Abstract: Efficient methods of stacking genes into plant genomes are needed to expedite transfer of multigenic traits into diverse crops grown in a variety of environments. Over two decades of research has identified several site-specific recombinases that carry out efficient cis and trans recombination between their recognition sites artificially introduced into plant chromosomes. The specificity and efficiency of recombinases make them extremely attractive for genome engineering applications, including transgene stacking. In plant biotechnology, recombinases have mostly been used for removing selectable marker genes, but they have the potential for much more complex engineering. Until recently, reversibility of recombination, a property of the tyrosine family of recombinases, had limited their use in gene stacking because it requires successive rounds of transformation for integrating genes into the engineered sites. However, key advances in the field - including the use of unidirectional recombination systems and modifications of recombinase recognition target sites -have overcome these challenges and paved the way for repeated site-specific integrations into the selected site. The ability to do gene stacking is vital for improvement of important crops because most traits of value are controlled by multiple genes, which could be delivered as a single genetic package by crossing the transgenic plants to elite varieties. Recombinases, by virtue of their specificity and efficiency in plant cells, emerge as powerful tools for a variety of applications including gene stacking.

Last Modified: 10/17/2017
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