Engineering Designer Transcription Activator‐‐Like Effector Nucleases (TALENs) by REAL or REAL‐Fast Assembly

摘要: 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, pluripotent stem cells. Here we describe detailed protocol the serial, hierarchical assembly TALENs that requires neither PCR nor specialized multi-fragment ligations can be implemented by any laboratory. This restriction enzyme ligation (REAL) practiced using small plasmid library user-friendly, web-based software both identifies target sites sequences interest generates printable graphical guides facilitate TALENs. All plasmids required to perform REAL method publicly available through Addgene distribution service (http://www.addgene.org/talengineering). Alternatively, decrease time effort required, users practice encoding pre-assembled TALE repeats. With platform reagents, protocols, describe, researchers easily engineer multiple within two weeks or less standard cloning techniques.