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

Satoshi Ansai, Keiji Inohaya, Yasutoshi Yoshiura, Manfred Schartl, Norihito Uemura, Ryosuke Takahashi, Masato Kinoshita

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)98-107
Number of pages10
JournalDevelopment Growth and Differentiation
Volume56
Issue number1
DOIs
Publication statusPublished - 2014 Jan

Keywords

  • Heteroduplex mobility assay
  • Medaka
  • Microhomology
  • Targeted mutagenesis
  • Transcription activator-like effector nuclease

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology

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