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Isolation of single-base genome-edited human iPS cells without antibiotic selection

Nature Methods volume 11, pages 291–293 (2014)Cite this article

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Abstract

Precise editing of human genomes in pluripotent stem cells by homology-driven repair of targeted nuclease–induced cleavage has been hindered by the difficulty of isolating rare clones. We developed an efficient method to capture rare mutational events, enabling isolation of mutant lines with single-base substitutions without antibiotic selection. This method facilitates efficient induction or reversion of mutations associated with human disease in isogenic human induced pluripotent stem cells.

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Figure 1: ddPCR-based detection of a point mutation in human iPS cells.
Figure 2: Point mutagenesis in human iPS cells.

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Acknowledgements

We thank members of the Conklin laboratory for technical assistance and critical reading of the manuscript, B.G. Bruneau, D. Srivastava, S. Yamanaka, K. Tomoda, Y. Hayashi, K.E. Eilertson, B.S. Moriarity, D.A. Largaespada and W.A. Weiss for valuable discussions and advice, A.K. Holloway for generating the list of unique CRISPR target sites, K. Carver-Moore for her technical assistance in iPS cell culture on 96-well plates, M. Porteus and M. Rahdar (Stanford University) for providing us with the TALEN backbone plasmid MR015, and members of the Roddenberry Stem Cell Core at Gladstone Institutes for providing a stimulating environment. Y.M. is a recipient of a Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad and the Uehara Memorial Foundation Research Fellowship. L.M.J. is supported by a postdoctoral fellowship, TG2-01160, from the California Institute of Regenerative Medicine. M.H. is supported by a postdoctoral fellowship, PF-13-295-01–TBG, from the American Cancer Society. B.R.C. received support from the US National Heart, Lung, and Blood Institute, National Institutes of Health, U01-HL100406, U01-GM09614, R01-HL108677, U01-HL098179 and R01-HL060664.

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Authors and Affiliations

  1. Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA

    Yuichiro Miyaoka, Amanda H Chan, Luke M Judge, Jennie Yoo, Trieu D Nguyen, Paweena P Lizarraga, Po-Lin So & Bruce R Conklin

  2. Department of Pediatrics, University of California San Francisco, San Francisco, California, USA

    Luke M Judge

  3. Department of Neurology, University of California San Francisco, San Francisco, California, USA

    Miller Huang

  4. Department of Medicine, University of California San Francisco, San Francisco, California, USA

    Bruce R Conklin

  5. Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California, USA

    Bruce R Conklin

Authors
  1. Yuichiro Miyaoka
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  2. Amanda H Chan
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  3. Luke M Judge
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  4. Jennie Yoo
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  5. Miller Huang
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  6. Trieu D Nguyen
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  7. Paweena P Lizarraga
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  8. Po-Lin So
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  9. Bruce R Conklin
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Contributions

Y.M. and B.R.C. conceived and designed the experiments. Y.M. and A.H.C. conducted most of the experiments. L.M.J. and J.Y. performed the BAG3 mutagenesis, and J.Y. constructed the PKP2 TALENs. M.H. designed and constructed the PHOX2B TALENs and helped with the Surveyor assays. T.D.N. helped to conceive ddPCR experiments and to construct TALENs. P.P.L. conducted immunofluorescence staining. Y.M., P.-L.S. and B.R.C. wrote the manuscript with support from all authors.

Corresponding author

Correspondence to Bruce R Conklin.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, and Supplementary Tables 1, 2, 4 and 5 (PDF 3062 kb)

Supplementary Table 3

On- and off-target sites for the PHOX2B and PRKAG2 TALENs predicted by TALENoffer (XLSX 59 kb)

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Miyaoka, Y., Chan, A., Judge, L. et al. Isolation of single-base genome-edited human iPS cells without antibiotic selection. Nat Methods 11, 291–293 (2014). https://doi.org/10.1038/nmeth.2840

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