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

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

Author information

1
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo, Kyoto, 606-8502, Japan.

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.

KEYWORDS:

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

PMID:
24286287
DOI:
10.1111/dgd.12104
[Indexed for MEDLINE]

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