4.8 Article

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

期刊

NUCLEIC ACIDS RESEARCH
卷 40, 期 16, 页码 8001-8010

出版社

OXFORD UNIV PRESS
DOI: 10.1093/nar/gks518

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资金

  1. National Institutes of Health (NIH) [R01 GM088040, DP1 OD006862, T32 CA009216, K01 AG031300]
  2. Jim and Ann Orr MGH Research Scholar award
  3. Charles and Ann Sanders MGH Research Scholar award
  4. National Science Foundation [DBI-0923827]

向作者/读者索取更多资源

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.

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