Engineering Designer Transcription Activator‐‐Like Effector Nucleases (TALENs) by REAL or REAL‐Fast Assembly
2012; Wiley; Volume: 100; Issue: 1 Linguagem: Inglês
10.1002/0471142727.mb1215s100
ISSN1934-3639
AutoresDeepak Reyon, Cyd Khayter, Maureen Regan, J. Keith Joung, Jeffry D. Sander,
Tópico(s)RNA and protein synthesis mechanisms
ResumoAbstract 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 r estriction e nzyme a nd l igation (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.
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