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

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)363-367
Number of pages5
JournalBiology Open
Volume2
Issue number4
DOIs
Publication statusPublished - 2013 Apr 15

Keywords

  • Genome editing
  • LacZ blue-white selection
  • Quantitative assay
  • TALEN

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

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  • Cite this

    Hisano, Y., Ota, S., Arakawa, K., Muraki, M., Kono, N., Oshita, K., Sakuma, T., Tomita, M., Yamamoto, T., Okada, Y., & Kawahara, A. (2013). Quantitative assay for TALEN activity at endogenous genomic loci. Biology Open, 2(4), 363-367. https://doi.org/10.1242/bio.20133871