Heterodimeric TALENs induce targeted heritable mutations in the crustacean Daphnia magna
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 o...
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doaj-e6c65550614d4ef598d87f404268d3db2021-06-02T15:44:10ZengThe Company of BiologistsBiology Open2046-63902015-02-014336436910.1242/bio.2014973820149738Heterodimeric TALENs induce targeted heritable mutations in the crustacean Daphnia magnaAkiko Naitou0Yasuhiko Kato1Takashi Nakanishi2Tomoaki Matsuura3Hajime Watanabe4 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan 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.http://bio.biologists.org/content/4/3/364Heterodimeric TALENsHeritable mutationBiallelic mutationDaphnia magnaEyelessDsRed2 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Akiko Naitou Yasuhiko Kato Takashi Nakanishi Tomoaki Matsuura Hajime Watanabe |
spellingShingle |
Akiko Naitou Yasuhiko Kato Takashi Nakanishi Tomoaki Matsuura Hajime Watanabe Heterodimeric TALENs induce targeted heritable mutations in the crustacean Daphnia magna Biology Open Heterodimeric TALENs Heritable mutation Biallelic mutation Daphnia magna Eyeless DsRed2 |
author_facet |
Akiko Naitou Yasuhiko Kato Takashi Nakanishi Tomoaki Matsuura Hajime Watanabe |
author_sort |
Akiko Naitou |
title |
Heterodimeric TALENs induce targeted heritable mutations in the crustacean Daphnia magna |
title_short |
Heterodimeric TALENs induce targeted heritable mutations in the crustacean Daphnia magna |
title_full |
Heterodimeric TALENs induce targeted heritable mutations in the crustacean Daphnia magna |
title_fullStr |
Heterodimeric TALENs induce targeted heritable mutations in the crustacean Daphnia magna |
title_full_unstemmed |
Heterodimeric TALENs induce targeted heritable mutations in the crustacean Daphnia magna |
title_sort |
heterodimeric talens induce targeted heritable mutations in the crustacean daphnia magna |
publisher |
The Company of Biologists |
series |
Biology Open |
issn |
2046-6390 |
publishDate |
2015-02-01 |
description |
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. |
topic |
Heterodimeric TALENs Heritable mutation Biallelic mutation Daphnia magna Eyeless DsRed2 |
url |
http://bio.biologists.org/content/4/3/364 |
work_keys_str_mv |
AT akikonaitou heterodimerictalensinducetargetedheritablemutationsinthecrustaceandaphniamagna AT yasuhikokato heterodimerictalensinducetargetedheritablemutationsinthecrustaceandaphniamagna AT takashinakanishi heterodimerictalensinducetargetedheritablemutationsinthecrustaceandaphniamagna AT tomoakimatsuura heterodimerictalensinducetargetedheritablemutationsinthecrustaceandaphniamagna AT hajimewatanabe heterodimerictalensinducetargetedheritablemutationsinthecrustaceandaphniamagna |
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