Design, evaluation, and screening methods for efficient targeted mutagenesis with transcription activator‐like effector nucleases in medaka. (28th November 2013)

• Record Type: Journal Article
• Authors: Ansai, Satoshi; Inohaya, Keiji; Yoshiura, Yasutoshi; Schartl, Manfred; Uemura, Norihito; Takahashi, Ryosuke; Kinoshita, Masato
• Is Part Of: Development growth and differentiation. Volume 56:Number 1(2014)
• Page Start: 98
• Page End: 107
• Publication Date: 2013-11-28
• Subjects: Embryology -- Periodicals; Developmental biology -- Periodicals; Growth -- Periodicals; 574.3
• DOI: 10.1111/dgd.12104
• Languages: English
• ISSNs: 0012-1592

Abstract

<abstract abstract-type="main" id="dgd12104-abs-0001"> <title> <x xml:space="preserve">Abstract</x> </title> <p>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, <italic>Oryzias latipes</italic>. We performed a heteroduplex mobility assay (HMA) using an automated microchip electrophoresis system, which is a simple and high‐throughput method for evaluation of <italic>in vivo</italic> 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.</p> </abstract>