title: Engineering designer transcription activator-like effector nucleases (TALENs) by REAL or REAL-Fast assembly.
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.
authors.affiliation: Molecular Pathology Unit, Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
authors.forename: Deepak
authors.initials: D
authors.lastname: Reyon
authors.forename: Cyd
authors.initials: C
authors.lastname: Khayter
authors.forename: Maureen R
authors.initials: MR
authors.lastname: Regan
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
authors.forename: Jeffry D
authors.initials: JD
authors.lastname: Sander
mesh_terms: D000818:Animals; D011088:DNA Ligases / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D010944:Plants; D011995:Recombination, Genetic; D015533:Transcriptional Activation
chemical_list: D003851:Deoxyribonucleases; D004262:DNA Restriction Enzymes; D011088:DNA Ligases