Quantitative assay for TALEN activity at endogenous genomic loci
Yu Hisano,
Satoshi Ota,
Kazuharu Arakawa,
Michiko Muraki,
Nobuaki Kono,
Kazuki Oshita,
Tetsushi Sakuma,
Masaru Tomita,
Takashi Yamamoto,
Yasushi Okada,
Atsuo Kawahara
Affiliations
Yu Hisano
Laboratory for Cardiovascular Molecular Dynamics, Quantitative Biology Center, RIKEN, Furuedai 6-2-3, Suita, Osaka 565-0874, Japan
Satoshi Ota
Laboratory for Cardiovascular Molecular Dynamics, Quantitative Biology Center, RIKEN, Furuedai 6-2-3, Suita, Osaka 565-0874, Japan
Kazuharu Arakawa
Institute for Advanced Biosciences, Keio University, Fujisawa, Kanagawa, 252-0882, Japan
Michiko Muraki
Laboratory for Cardiovascular Molecular Dynamics, Quantitative Biology Center, RIKEN, Furuedai 6-2-3, Suita, Osaka 565-0874, Japan
Nobuaki Kono
Institute for Advanced Biosciences, Keio University, Fujisawa, Kanagawa, 252-0882, Japan
Kazuki Oshita
Institute for Advanced Biosciences, Keio University, Fujisawa, Kanagawa, 252-0882, Japan
Tetsushi Sakuma
Molecular Genetics Laboratory, Department of Mathematical and Life Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima, Hiroshima 739-8526, Japan
Masaru Tomita
Institute for Advanced Biosciences, Keio University, Fujisawa, Kanagawa, 252-0882, Japan
Takashi Yamamoto
Molecular Genetics Laboratory, Department of Mathematical and Life Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima, Hiroshima 739-8526, Japan
Yasushi Okada
Laboratory for Cell Polarity Regulation, Quantitative Biology Center, RIKEN, Furuedai 6-2-3, Suita, Osaka 565-0874, Japan
Atsuo Kawahara
Laboratory for Cardiovascular Molecular Dynamics, Quantitative Biology Center, RIKEN, Furuedai 6-2-3, Suita, Osaka 565-0874, Japan
Summary Artificially designed nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can induce a targeted DNA double-strand break at the specific target genomic locus, leading to the frameshift-mediated gene disruption. However, the assays for their activity on the endogenous genomic loci remain limited. Herein, we describe a versatile modified lacZ assay to detect frameshifts in the nuclease target site. Short fragments of the genome DNA at the target or putative off-target loci were amplified from the genomic DNA of TALEN-treated or control embryos, and were inserted into the lacZα sequence for the conventional blue–white selection. The frequency of the frameshifts in the fragment can be estimated from the numbers of blue and white colonies. Insertions and/or deletions were easily determined by sequencing the plasmid DNAs recovered from the positive colonies. Our technique should offer broad application to the artificial nucleases for genome editing in various types of model organisms.