Design, evaluation, and screening methods for efficient targeted mutagenesis with transcription activator-like effector nucleases in medaka

Dev Growth Differ. 2014 Jan;56(1):98-107. doi: 10.1111/dgd.12104. Epub 2013 Nov 28.


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.

Keywords: heteroduplex mobility assay; medaka; microhomology; targeted mutagenesis; transcription activator-like effector nuclease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism*
  • Gene Targeting / methods*
  • Heteroduplex Analysis
  • Mutagenesis, Site-Directed / methods*
  • Oryzias / genetics*


  • Endodeoxyribonucleases