title: Design, evaluation, and screening methods for efficient targeted mutagenesis with transcription activator-like effector nucleases in medaka.
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.
authors.affiliation: Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo, Kyoto, 606-8502, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ansai
authors.forename: Keiji
authors.initials: K
authors.lastname: Inohaya
authors.forename: Yasutoshi
authors.initials: Y
authors.lastname: Yoshiura
authors.forename: Manfred
authors.initials: M
authors.lastname: Schartl
authors.forename: Norihito
authors.initials: N
authors.lastname: Uemura
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Takahashi
authors.forename: Masato
authors.initials: M
authors.lastname: Kinoshita
mesh_terms: D000818:Animals; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D020180:Heteroduplex Analysis; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009990:Oryzias / Q000235:genetics*
chemical_list: D004706:Endodeoxyribonucleases
keywords: heteroduplex mobility assay; medaka; microhomology; targeted mutagenesis; transcription activator-like effector nuclease