title: Heterodimeric TALENs induce targeted heritable mutations in the crustacean Daphnia magna.
abstract: Transcription activator-like effector nucleases (TALENs) are artificial nucleases harboring a customizable DNA-binding domain and a FokI nuclease domain. The high specificity of the DNA-binding domain and the ease of design have enabled researchers to use TALENs for targeted mutagenesis in various organisms. Here, we report the development of TALEN-dependent targeted gene disruption in the crustacean Daphnia magna, the emerging model for ecological and toxicological genomics. First, a reporter transgene DsRed2 (EF1α-1::DsRed2) was targeted. Using the Golden Gate method with a GoldyTALEN scaffold, we constructed homodimeric and heterodimeric TALENs containing wild-type and ELD/KKR FokI domains. mRNAs that coded for either the customized homodimeric or heterodimeric TALENs were injected into one-cell-stage embryos. The high mortality of embryos injected with homodimeric TALEN mRNAs prevented us from detecting mutations. In contrast, embryos injected with heterodimeric TALEN mRNAs survived and 78%-87% of the adults lost DsRed2 fluorescence in a large portion of cells throughout the body. In addition, these adults produced non-fluorescent progenies, all of which carried mutations at the dsRed2 locus. We also tested heterodimeric TALENs targeted for the endogenous eyeless gene and found that biallelic mutations could be transmitted through germ line cells at a rate of up to 22%. Both somatic and heritable mutagenesis efficiencies of TALENs were higher than those of the CRISPR/Cas9 system that we recently developed. These results suggest that the TALEN system may efficiently induce heritable mutations into the target genes, which will further contribute to the progress of functional genomics in D. magna.
authors: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors: Akiko
authors: A
authors: Naitou
authors: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Frontier Research Base for Global Young Researchers, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors: Yasuhiko
authors: Y
authors: Kato
authors: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors: Takashi
authors: T
authors: Nakanishi
authors: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors: Tomoaki
authors: T
authors: Matsuura
authors: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan watanabe@bio.eng.osaka-u.ac.jp.
authors: Hajime
authors: H
authors: Watanabe
keywords: Biallelic mutation; Daphnia magna; DsRed2; Eyeless; Heritable mutation; Heterodimeric TALENs