Targeted mutagenesis of genes-of-interest, or gene-knockout, is a powerful method to address the functions of genes. Engineered nucleases have enabled this approach in various organisms because of their ease of use. The ascidian Ciona intestinalis is an excellent organism to analyze gene functions by means of genetic technologies. In our previous study, we reported mutagenesis of Ciona somatic cells with TALE nucleases (TALENs) by electroporating expression constructs. In this study, we report germ cell mutagenesis of Ciona by microinjecting mRNAs encoding TALENs. TALEN mRNAs introduced mutations to target genes in both somatic and germ cells. TALEN-mediated mutations in the germ cell genome were inherited by the next generation. We conclude that knockout lines of Ciona that have disrupted target genes can be established through TALEN-mediated germ cell mutagenesis.