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 a detailed protocol for the serial, hierarchical assembly of TALENs that requires neither PCR nor specialized multi-fragment ligations and that can be implemented by any laboratory. This restriction enzyme and ligation (REAL) protocol can be practiced using a small plasmid library 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. All plasmids required to perform the REAL assembly method are publicly available through the Addgene plasmid distribution service (http://www.addgene.org/talengineering). Alternatively, to decrease the time and effort required, users can practice REAL using a library of plasmids encoding pre-assembled TALE repeats. With the platform of reagents, protocols, and software we describe, researchers can easily engineer multiple TALENs within two weeks or less using standard cloning techniques.
Keywords: TALEN, TALENs, engineered TAL nucleases, engineered TALE nucleases, REAL, REAL-Fast, protein engineering, DNA-binding domains, FLASH