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ZFIN ID: ZDB-PUB-120612-9
Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs
Cade, L., Reyon, D., Hwang, W.Y., Tsai, S.Q., Patel, S., Khayter, C., Joung, J.K., Sander, J.D., Peterson, R.T., and Yeh, J.R.
Date: 2012
Source: Nucleic acids research   40(16): 8001-8010 (Journal)
Registered Authors: Peterson, Randall, Yeh, Jing-Ruey (Joanna)
Keywords: none
MeSH Terms:
  • Alleles
  • Animals
  • Base Sequence
  • DNA-Binding Proteins/chemistry
  • Deoxyribonucleases, Type II Site-Specific/chemistry*
  • Deoxyribonucleases, Type II Site-Specific/metabolism*
  • Dimerization
  • Gene Knockout Techniques*
  • Molecular Sequence Data
  • Mutagenesis
  • Mutation*
  • Trans-Activators/chemistry
  • Zebrafish/genetics*
PubMed: 22684503 Full text @ Nucleic Acids Res.

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.