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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR TOOLS TO MINIMIZE RISK IN GENETICALLY ENGINEERED CROPS

Location: Crop Improvement & Utilization Research

Title: ParA resolvase catalyzes site-specific excision of DNA from the Arabidopsis genome

Authors
item Thomson, James
item Yuan-Yeu, Yau - UC BERKELEY
item Blanvillain, Robert - UC BERKELEY
item Nunes, Wylla -
item Chiniquy, Dawn -
item Thilmony, Roger
item Ow, David

Submitted to: Transgenic Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 30, 2008
Publication Date: April 18, 2009
Citation: Thomson, J.G., Yuan-Yeu, Y., Blanvillain, R., Chiniquy, D., Thilmony, R.L., Ow, D.W. 2009. ParA resolvase catalyzes site-specific excision of DNA from the Arabidopsis genome. Transgenic Research. 18:237-248.

Interpretive Summary: Site-specific recombinases are enzymes that are capable of removing unwanted antibiotic or herbicide genes used as selectable markers during genetic engineering prior to public marketing. The conservative nature of the enzymes does not add or delete nucleotides thereby maintaining transgene integrity. Currently most useful recombinase systems are patented by companies and do not allow freedom to operate without first purchasing a license, if one is available. To help facilitate the freedom to operate and stimulate development of this technology, our lab has developed and tested a number of novel recombinases to better control genomic engineering in plants. We report here evidence that the novel parA recombinase can precisely remove DNA from the Arabidopsis genome. We further show that the genomic deletion event can be passed through the germ-line and inherited independently from the parA recombinase gene.

Technical Abstract: The small serine resolvase ParA from bacterial plasmids RK2 and RP4 catalyzes the recombination of two identical 133 bp recombination sites known as MRS. Previously, we reported that ParA is active in the fission yeast Schizosaccharomyces pombe. In this work, the parA recombinase gene was placed under the control of the Arabidopsis OXS3 promoter and introduced into Arabidopsis lines harboring a chromosomally integrated MRS-flanked target. The ParA recombinase excised the MRS-flanked DNA and the excision event was detected in subsequent generations in the absence of ParA, indicating germinal transmission of the excision event. The precise site-specific deletion by the ParA recombination system in planta demonstrates that the ParA recombinase can be used to remove transgenic DNA, such as selectable markers or other introduced transgenes that are no longer desired in the final product.

Last Modified: 9/23/2014
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