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Genome editing using engineered nucleases such as transcription activator-like effector nucleases ( TALENs) has become a powerful technology for reverse genetics. In this study, we have described efficient detection methods for TALEN-induced mutations at endogenous loci and presented guidelines of TALEN design for efficient targeted mutagenesis in medaka, Oryzias latipes. We performed a heteroduplex mobility assay ( HMA) using an automated microchip electrophoresis system, which is a simple and high-throughput method for evaluation of in vivo activity of TALENs and for genotyping mutant fish of F1 or later generations. We found that a specific pattern of mutations is dominant for TALENs harboring several base pairs of homologous sequences in target sequence. Furthermore, we found that a 5′ T, upstream of each TALEN-binding sequence, is not essential for genomic DNA cleavage. Our findings provide information that expands the potential of TALENs and other engineered nucleases as tools for targeted genome editing in a wide range of organisms, including medaka. [ABSTRACT FROM AUTHOR]