PUBLICATION

Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs)

Authors
Moore, F.E., Reyon, D., Sander, J.D., Martinez, S.A., Blackburn, J.S., Khayter, C., Ramirez, C.L., Joung, J.K., and Langenau, D.M.
ID
ZDB-PUB-120604-11
Date
2012
Source
PLoS One   7(5): e37877 (Journal)
Registered Authors
Langenau, David
Keywords
none
MeSH Terms
  • Animals
  • Base Sequence
  • DNA/genetics*
  • DNA/metabolism
  • Deoxyribonucleases/genetics
  • Deoxyribonucleases/metabolism*
  • Deoxyribonucleases, Type II Site-Specific/genetics
  • Deoxyribonucleases, Type II Site-Specific/metabolism
  • Endonucleases/genetics
  • Endonucleases/metabolism*
  • Molecular Sequence Data
  • Mutagenesis*
  • Mutation Rate
  • Two-Hybrid System Techniques
  • Zebrafish/embryology
  • Zebrafish/genetics*
  • Zebrafish/metabolism
  • Zebrafish Proteins/genetics
  • Zebrafish Proteins/metabolism*
  • Zinc Fingers
PubMed
22655075 Full text @ PLoS One
Abstract

Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%–76.8% compared to 1.1%–3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

Genes / Markers
Figures
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Expression
Phenotype
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping
Errata and Notes