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

29 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
Externally publishedYes

Fingerprint

Ciona intestinalis
Chordata
Zinc Fingers
Mutagenesis
Genome
Mutation
Embryonic Development
Double-Stranded DNA Breaks
Homologous Recombination
Mutation Rate
Growth and Development

Keywords

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

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology

Cite this

Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases. / Kawai, Narudo; Ochiai, Hiroshi; Sakuma, Tetsushi; Yamada, Lixy; Sawada, Hitoshi; Yamamoto, Takashi; Sasakura, Yasunori.

In: Development Growth and Differentiation, Vol. 54, No. 5, 06.2012, p. 535-545.

Research output: Contribution to journalArticle

Kawai, Narudo ; Ochiai, Hiroshi ; Sakuma, Tetsushi ; Yamada, Lixy ; Sawada, Hitoshi ; Yamamoto, Takashi ; Sasakura, Yasunori. / Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases. In: Development Growth and Differentiation. 2012 ; Vol. 54, No. 5. pp. 535-545.
@article{784de04f98d943158fa60c99140de34f,
title = "Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases",
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.",
keywords = "Ascidian, Ciona intestinalis, Enhanced green fluorescent protein, Gene targeting, Zinc-finger nuclease",
author = "Narudo Kawai and Hiroshi Ochiai and Tetsushi Sakuma and Lixy Yamada and Hitoshi Sawada and Takashi Yamamoto and Yasunori Sasakura",
year = "2012",
month = "6",
doi = "10.1111/j.1440-169X.2012.01355.x",
language = "English",
volume = "54",
pages = "535--545",
journal = "Development, growth & differentiation",
issn = "0012-1592",
publisher = "Wiley-Blackwell",
number = "5",

}

TY - JOUR

T1 - Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases

AU - Kawai, Narudo

AU - Ochiai, Hiroshi

AU - Sakuma, Tetsushi

AU - Yamada, Lixy

AU - Sawada, Hitoshi

AU - Yamamoto, Takashi

AU - Sasakura, Yasunori

PY - 2012/6

Y1 - 2012/6

N2 - 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.

AB - 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.

KW - Ascidian

KW - Ciona intestinalis

KW - Enhanced green fluorescent protein

KW - Gene targeting

KW - Zinc-finger nuclease

UR - http://www.scopus.com/inward/record.url?scp=84862694393&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84862694393&partnerID=8YFLogxK

U2 - 10.1111/j.1440-169X.2012.01355.x

DO - 10.1111/j.1440-169X.2012.01355.x

M3 - Article

VL - 54

SP - 535

EP - 545

JO - Development, growth & differentiation

JF - Development, growth & differentiation

SN - 0012-1592

IS - 5

ER -