Unit

UNIT 12.15 Engineering Designer Transcription Activator--Like Effector Nucleases (TALENs) by REAL or REAL-Fast Assembly

  1. Deepak Reyon1,2,
  2. Cyd Khayter1,
  3. Maureen R. Regan1,
  4. J. Keith Joung1,2,
  5. Jeffry D. Sander1,2

Published Online: 1 OCT 2012

DOI: 10.1002/0471142727.mb1215s100

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Reyon, D., Khayter, C., Regan, M. R., Joung, J. K. and Sander, J. D. 2012. Engineering Designer Transcription Activator--Like Effector Nucleases (TALENs) by REAL or REAL-Fast Assembly. Current Protocols in Molecular Biology. 100:12.15:12.15.1–12.15.14.

Author Information

  1. 1

    Molecular Pathology Unit, Center for Computational and Integrative Biology and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts

  2. 2

    Department of Pathology, Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 OCT 2012
  2. Published Print: OCT 2012

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. Curr. Protoc. Mol. Biol. 100:12.15.1-12.15.14. © 2012 by John Wiley & Sons, Inc.

Keywords:

  • TALEN;
  • TALENs;
  • engineered TAL effector nucleases;
  • engineered TALE nucleases;
  • REAL;
  • REAL-Fast;
  • protein engineering;
  • DNA-binding domains;
  • FLASH