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G3 (Bethesda). 2013 Oct 3;3(10):1707-15. doi: 10.1534/g3.113.006270.

Targeted deletion and inversion of tandemly arrayed genes in Arabidopsis thaliana using zinc finger nucleases.

Author information

1
Department of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455.

Abstract

Tandemly arrayed genes (TAGs) or gene clusters are prevalent in higher eukaryotic genomes. For example, approximately 17% of genes are organized in tandem in the model plant Arabidopsis thaliana. The genetic redundancy created by TAGs presents a challenge for reverse genetics. As molecular scissors, engineered zinc finger nucleases (ZFNs) make DNA double-strand breaks in a sequence-specific manner. ZFNs thus provide a means to delete TAGs by creating two double-strand breaks in the gene cluster. Using engineered ZFNs, we successfully targeted seven genes from three TAGs on two Arabidopsis chromosomes, including the well-known RPP4 gene cluster, which contains eight resistance (R) genes. The resulting gene cluster deletions ranged from a few kb to 55 kb with frequencies approximating 1% in somatic cells. We also obtained large chromosomal deletions of ~9 Mb at approximately one tenth the frequency, and gene cluster inversions and duplications also were achieved. This study demonstrates the ability to use sequence-specific nucleases in plants to make targeted chromosome rearrangements and create novel chimeric genes for reverse genetics and biotechnology.

KEYWORDS:

Arabidopsis; deletion; inversion; tandemly arrayed genes (TAGs); zinc finger nuclease (ZFN)

PMID:
23979943
PMCID:
PMC3789795
DOI:
10.1534/g3.113.006270
[Indexed for MEDLINE]
Free PMC Article

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