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Curr Protoc Mol Biol. 2012 Oct;Chapter 12:Unit 12.15. doi: 10.1002/0471142727.mb1215s100.

Engineering designer transcription activator-like effector nucleases (TALENs) by REAL or REAL-Fast assembly.

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1
Molecular Pathology Unit, Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

Abstract

Engineered transcription activator-like effector nucleases (TALENs) are broadly useful tools for performing targeted genome editing in a wide variety of organisms and cell types including plants, zebrafish, C. elegans, rat, human somatic cells, and human pluripotent stem cells. Here we describe detailed protocols for the serial, hierarchical assembly of TALENs that require neither PCR nor specialized multi-fragment ligations and that can be implemented by any laboratory. These restriction enzyme and ligation (REAL)-based protocols can be practiced using plasmid libraries and user-friendly, Web-based software that both identifies target sites in sequences of interest and generates printable graphical guides that facilitate assembly of TALENs. With the described platform of reagents, protocols, and software, researchers can easily engineer multiple TALENs within 2 weeks using standard cloning techniques.

PMID:
23026907
PMCID:
PMC3753116
DOI:
10.1002/0471142727.mb1215s100
[Indexed for MEDLINE]
Free PMC Article
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