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Nucleic Acids Res DOI:10.1093/nar/gks518

Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs.

Publication TypeJournal Article
Year of Publication2012
AuthorsCade, L, Reyon, D, Hwang, WY, Tsai, SQ, Patel, S, Khayter, C, J Joung, K, Sander, JD, Peterson, RT, Yeh, J-RJoanna
JournalNucleic Acids Res
Date Published2012 Sep
KeywordsAlleles, Animals, Base Sequence, Deoxyribonucleases, Type II Site-Specific, Dimerization, DNA-Binding Proteins, Gene Knockout Techniques, Molecular Sequence Data, Mutagenesis, Mutation, Trans-Activators, Zebrafish

Transcription activator-like effector nucleases (TALENs) are powerful new research tools that enable targeted gene disruption in a wide variety of model organisms. Recent work has shown that TALENs can induce mutations in endogenous zebrafish genes, but to date only four genes have been altered, and larger-scale tests of the success rate, mutation efficiencies and germline transmission rates have not been described. Here, we constructed homodimeric TALENs to 10 different targets in various endogenous zebrafish genes and found that 7 nuclease pairs induced targeted indel mutations with high efficiencies ranging from 2 to 76%. We also tested obligate heterodimeric TALENs and found that these nucleases induce mutations with comparable or higher frequencies and have better toxicity profiles than their homodimeric counterparts. Importantly, mutations induced by both homodimeric and heterodimeric TALENs are passed efficiently through the germline, in some cases reaching 100% transmission. For one target gene sequence, we observed substantially reduced mutagenesis efficiency for a variant site bearing two mismatched nucleotides, raising the possibility that TALENs might be used to perform allele-specific gene disruption. Our results suggest that construction of one to two heterodimeric TALEN pairs for any given gene will, in most cases, enable researchers to rapidly generate knockout zebrafish.


Alternate JournalNucleic Acids Res.
PubMed ID22684503
PubMed Central IDPMC3439908
Grant ListDP1 GM105378 / GM / NIGMS NIH HHS / United States
R01 CA140188 / CA / NCI NIH HHS / United States
T32 CA009216 / CA / NCI NIH HHS / United States
R01 GM088040 / GM / NIGMS NIH HHS / United States
F32 GM105189 / GM / NIGMS NIH HHS / United States
K01 AG031300 / AG / NIA NIH HHS / United States
DP1 OD006862 / OD / NIH HHS / United States