UNIT 12.15 Engineering Designer Transcription Activator--Like Effector Nucleases (TALENs) by REAL or REAL-Fast Assembly
Published Online: 1 OCT 2012
Copyright © 2012 by John Wiley & Sons, Inc.
Lab Protocol Title
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.
- Published Online: 1 OCT 2012
- Published Print: OCT 2012
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.
- engineered TAL effector nucleases;
- engineered TALE nucleases;
- protein engineering;
- DNA-binding domains;