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J Integr Plant Biol. 2013 Nov;55(11):1092-103. doi: 10.1111/jipb.12091. Epub 2013 Sep 18.

Site-specific gene targeting using transcription activator-like effector (TALE)-based nuclease in Brassica oleracea.

Author information

  • 1Key Laboratory of Horticulture Science for Southern Mountainous Regions, Southwest University, Chongqing, 400715, China.

Abstract

Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors (TALEs) contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases (TALENs), constructed using a method called "unit assembly", specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non-homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.

© 2013 Institute of Botany, Chinese Academy of Sciences.

KEYWORDS:

Brassica oleracea; FRIGIDA; double-strand break; gene targeting; non-homologous end-joining; type III transcription activator-like effector-based nucleases

PMID:
23870552
[PubMed - indexed for MEDLINE]
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