Abstract

The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease with a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications1,2,3,4,5. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show that this enhanced TALEN toolkit has a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino-based targeted gene knockdowns6. With this updated TALEN system, we successfully used single-stranded DNA oligonucleotides to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair, including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.

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Acknowledgements

State of Minnesota grant H001274506 to S.C.E. and D.F.V.; NIH GM63904 to S.C.E.; NIH grant P30DK084567 to S.C.E. and K.J.C.; NIH grant DK083219 to V.M.B.; Mayo Foundation; NIH DA032194 to K.J.C.; NIH grant R41HL108440 to D.F.C. and S.C.F.; NIH grant GM088424 to J.J.E.; NSF grant DBI0923827 to D.F.V.; General Research Fund (HKU771611, HKU771110, HKU769809M) from the Research Grant Council, The University of Hong Kong and the Tang King Yin Research Fund to A.C.M. and A.Y.H.L. We thank G. Davis for discussion on ssDNA use with custom restriction enzymes, H. J. Fadel for in vitro RNA synthesis, and S. Westcot for comments on this manuscript. We thank G. Moulder for help in DNA analyses and members of the Mayo Clinic Zebrafish Core Facility for excellent animal care.

Author information

Author notes

    • Victoria M. Bedell
    • , Ying Wang
    •  & Jarryd M. Campbell

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA

    • Victoria M. Bedell
    • , Jarryd M. Campbell
    • , Tanya L. Poshusta
    • , Randall G. Krug II
    • , Sumedha G. Penheiter
    • , Alvin C. Ma
    • , Karl J. Clark
    •  & Stephen C. Ekker
  2. Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa 50011, USA

    • Ying Wang
    •  & Jeffrey J. Essner
  3. Department of Genetics, Cell Biology and Development, the Center for Genome Engineering, and the Department of Animal Science, University of Minnesota, Minneapolis, Minnesota 55455, USA

    • Colby G. Starker
    • , Wenfang Tan
    • , Scott C. Fahrenkrug
    • , Daniel F. Carlson
    •  & Daniel F. Voytas
  4. Department of Medicine, LKS Faculty of Medicine, the University of Hong Kong, Pok Fu Lam Road, Hong Kong

    • Alvin C. Ma
    •  & Anskar Y. H. Leung
  5. Recombinetics, Inc., 2575 University Ave. West, Suite 100, Saint Paul, Minnesota 55114, USA

    • Scott C. Fahrenkrug
    •  & Daniel F. Carlson

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Contributions

W.T. designed and constructed full-length pT3TS-Tal vectors targeting Danio rerio crhr1 and crhr2 against exon sequences provided by K.J.C.; D.F.C. and S.C.F. designed and produced the pT3TS-Tal and GoldyTALEN cloning vectors. D.F.C. synthesized crhr1 and crhr2 TALEN mRNA from the pT3TS-Tal vectors. C.G.S. designed and assembled ponzr1 TALENs and transferred TAL repeats from original crhr1 and crhr2 TALEN vectors into GoldyTALEN. T.L.P. and K.J.C. did initial crhr1 TALEN microinjections. T.L.P. performed initial characterization of crhr1 mutagenesis efficiency by PCR and restriction fragment length polymorphism analysis. T.L.P. and K.J.C. microinjected crhr2 TALEN and loxP oligonucleotides. T.L.P. performed initial characterization by PCR demonstrating loxP integration. R.G.K. fully characterized efficiency and sequence of somatic loxP insertions in the crhr2 locus. K.J.C. screened adult fin clips for mloxP integrations into crhr2. T.L.P. screened F1 offspring for mloxP integrations into crhr2 and together with J.M.C. cloned and sequenced integration events. K.J.C. designed experiments associated for crhr1 and crhr2 modification. K.J.C. selected loxP mutant JTZ17 for integration. A.C.M. developed initial zebrafish genetic testing, TALEN cell-free assays, and ssDNA HDR protocols. S.G.P. conducted the cell-free TALEN endonuclease assay. A.Y.H.L. contributed to the design of initial TALEN and ssDNA HDR experiments. Y.W. and J.J.E. conducted biallelic conversion TALEN experiments in somatic and germline tests. D.F.V. and S.C.E. initiated the strategy to use custom restriction enzymes for genome editing in zebrafish. S.C.E. developed the plan for HDR targeting using ssDNAs, conducted overall project design and data analysis, and wrote the initial manuscript text. All authors contributed to manuscript composition. V.M.B. and J.M.C. conducted ponzr1 and crhr1 TALEN scaffold comparison experiments. V.M.B. and K.J.C. designed ssDNA oligonucleotides for HDR experiments. V.M.B. and J.M.C. injected and screened the EcoRV and mloxP HDR experiments at the ponzr1 locus. J.M.C. ran quantitative data assessments and statistical analyses. V.M.B. and J.M.C. made first drafts and legends of Figs 1, 3 and 5. V.M.B. and J.M.C. conducted fin biopsy analyses of the ponzr1 locus. V.M.B. completed the analysis of ponzr1 germline transmission with assistance from J.M.C.

Competing interests

S.C.F., J.J.E., K.J.C. and D.F.V. hold shares in Recombinetics, Inc., a company that uses TALENs for genome modification in large animals. D.F.V. is a listed inventor on a patent application titled “TAL effector-mediated DNA modification” that is co-owned by Iowa State University and the University of Minnesota, and has been licensed to Cellectis, a European biotechnology company.

Corresponding author

Correspondence to Stephen C. Ekker.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figures 1-11, Supplementary Table 1 and legends for Supplementary Movies 1-3.

Videos

  1. 1.

    Supplementary Movie 1

    This movie shows red blood cell movement through the blood vessels in wild type embryos.

  2. 2.

    Supplementary Movie 2

    This movie shows red blood cell movement through the blood vessels in cdh5 MO-injected embryos.

  3. 3.

    Supplementary Movie 3

    This movie shows red blood cell movement through the blood vessels in cdh5 GoldyTALEN-injected embryos.

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DOI

https://doi.org/10.1038/nature11537

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