In vivo genome editing using a high-efficiency TALEN system - Nature

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Author notes

1. Victoria M. Bedell, Ying Wang and Jarryd M. Campbell: These authors contributed equally to this work.

Authors and Affiliations

1. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, 55905, Minnesota, 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, 50011, Iowa, USA

   Ying Wang & Jeffrey J. Essner

3. Department of Genetics, and the Department of Animal Science, Cell Biology and Development, the Center for Genome Engineering, University of Minnesota, Minneapolis, 55455, Minnesota, 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

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.

Corresponding author

Correspondence to Stephen C. Ekker.

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.

Cite this article

Bedell, V., Wang, Y., Campbell, J. et al. In vivo genome editing using a high-efficiency TALEN system. Nature 491, 114–118 (2012). https://doi.org/10.1038/nature11537

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• Received: 03 May 2012

• Accepted: 06 September 2012

• Published: 23 September 2012

• Issue Date: 01 November 2012

• DOI: https://doi.org/10.1038/nature11537