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

<jats:p>Zinc‐finger nucleases (<jats:styled-content style="fixed-case">ZFNs</jats:styled-content>) are engineered nucleases that induce <jats:styled-content style="fixed-case">DNA</jats:styled-content> double‐strand breaks (<jats:styled-content style="fixed-case">DSBs</jats:styled-content>) 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 <jats:styled-content style="fixed-case">DSB</jats:styled-content> induced by <jats:styled-content style="fixed-case">ZFNs</jats:styled-content> is repaired by non‐homologous end joining (<jats:styled-content style="fixed-case">NHEJ</jats:styled-content>) or by homologous recombination (<jats:styled-content style="fixed-case">HR</jats:styled-content>) mechanisms. Non‐homologous end joining induces some errors because it is independent of a reference <jats:styled-content style="fixed-case">DNA</jats:styled-content> sequence. Through the <jats:styled-content style="fixed-case">NHEJ</jats:styled-content> mechanism, <jats:styled-content style="fixed-case">ZFNs</jats:styled-content> generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of <jats:styled-content style="fixed-case">ZFNs</jats:styled-content> in the basal chordate <jats:italic> <jats:styled-content style="fixed-case">C</jats:styled-content>iona intestinalis</jats:italic>. As the target of <jats:styled-content style="fixed-case">ZFNs</jats:styled-content>, we chose an <jats:styled-content style="fixed-case"> <jats:italic>enhanced green fluorescent protein (EGFP)</jats:italic> </jats:styled-content> gene artificially inserted in the <jats:italic> <jats:styled-content style="fixed-case">C</jats:styled-content>. intestinalis</jats:italic> genome because this locus is neutral for the development and growth of <jats:italic> <jats:styled-content style="fixed-case">C</jats:styled-content>. intestinalis</jats:italic>, and the efficiency of mutagenesis with <jats:styled-content style="fixed-case">ZFNs</jats:styled-content> can thus be determined without any bias. We introduced <jats:styled-content style="fixed-case"> <jats:italic>EGFP</jats:italic> </jats:styled-content>‐<jats:styled-content style="fixed-case">ZFN mRNA</jats:styled-content>s into the embryos of an <jats:styled-content style="fixed-case"> <jats:italic>EGFP</jats:italic> </jats:styled-content>‐transgenic line and observed the mutation frequency in the target site of <jats:styled-content style="fixed-case"> <jats:italic>EGFP</jats:italic> </jats:styled-content>. We also examined the effects of the <jats:styled-content style="fixed-case"> <jats:italic>EGFP</jats:italic> </jats:styled-content>‐<jats:styled-content style="fixed-case">ZFNs</jats:styled-content> at off‐target sites resembling the <jats:styled-content style="fixed-case"> <jats:italic>EGFP</jats:italic> </jats:styled-content> target sequence in the <jats:italic> <jats:styled-content style="fixed-case">C</jats:styled-content>. intestinalis</jats:italic> genome in order to examine the specificity of <jats:styled-content style="fixed-case">ZFNs</jats:styled-content>. We further investigated the influence of <jats:styled-content style="fixed-case">ZFNs</jats:styled-content> on embryogenesis, and showed that adequate amounts of <jats:styled-content style="fixed-case">ZFNs</jats:styled-content>, 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 <jats:styled-content style="fixed-case">ZFNs</jats:styled-content> will be a powerful technique in <jats:italic> <jats:styled-content style="fixed-case">C</jats:styled-content>. intestinalis</jats:italic>.</jats:p>