Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases

Narudo Kawai, Hiroshi Ochiai, Tetsushi Sakuma, Lixy Yamada, Hitoshi Sawada, Takashi Yamamoto, Yasunori Sasakura

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Zinc-finger nucleases (ZFNs) are engineered nucleases that induce DNA double-strand breaks (DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non-homologous end joining (NHEJ) or by homologous recombination (HR) mechanisms. Non-homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C. intestinalis genome because this locus is neutral for the development and growth of C. intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP -ZFN mRNAs into the embryos of an EGFP -transgenic line and observed the mutation frequency in the target site of EGFP. We also examined the effects of the EGFP -ZFNs at off-target sites resembling the EGFP target sequence in the C. intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on-target site with less effect on the off-target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C. intestinalis.

Original languageEnglish
Pages (from-to)535-545
Number of pages11
JournalDevelopment Growth and Differentiation
Volume54
Issue number5
DOIs
Publication statusPublished - 2012 Jun

Keywords

  • Ascidian
  • Ciona intestinalis
  • Enhanced green fluorescent protein
  • Gene targeting
  • Zinc-finger nuclease

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology

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