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

研究成果: Article査読

31 被引用数 (Scopus)


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 frameshiftmediated 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 lacZa 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.

ジャーナルBiology Open
出版ステータスPublished - 2013 4月 15

ASJC Scopus subject areas

  • 生化学、遺伝学、分子生物学一般
  • 農業および生物科学一般


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