A protocol for TALEN construction and gene targeting in zebrafish

TALEN (Transcription activator-like effector nuclease) is a newly developed family of artificially engineered sequence-specific endonucleases, which consists of a highly specific and repetitive DNA-binding domain derived from TALE (Transcription activator-like effector) and fused with the non-specific endonuclease domain of FokⅠ. TALENs have been reported to be able to induce site specific genome modification in quite a few species and in vitro cultured cells. Here, we introduced the principles for target site selection and confirmation and described a brief experimental protocol for the easy construction of customized TALENs using unit assembly (UA) method and also for generation of and screening for TALEN-mediated targeted zebrafish mutants through microinjection of TALEN mRNAs into zebrafish embryos. Theoretically, the principles and methods we described here are also applicable to gene targeting in other species.