Protocol | Published:

Simultaneous reprogramming and gene editing of human fibroblasts

Nature Protocols volume 13, pages 875898 (2018) | Download Citation

Abstract

The utility of human induced pluripotent stem cells (iPSCs) is enhanced by an ability to precisely modify a chosen locus with minimal impact on the remaining genome. However, the derivation of gene-edited iPSCs typically involves multiple steps requiring lengthy culture periods and several clonal events. Here, we describe a one-step protocol for reliable generation of clonally derived gene-edited iPSC lines from human fibroblasts in the absence of drug selection or FACS enrichment. Using enhanced episomal-based reprogramming and CRISPR/Cas9 systems, gene-edited and passage-matched unmodified iPSC lines are obtained following a single electroporation of human fibroblasts. To minimize unwanted mutations within the target locus, we use a Cas9 variant that is associated with decreased nonhomologous end-joining (NHEJ) activity. This protocol outlines in detail how this streamlined approach can be used for both monoallelic and biallelic introduction of specific base changes or transgene cassettes in a manner that is efficient, rapid (68 weeks), and cost-effective.

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Acknowledgements

We acknowledge A. Mallett, Royal Brisbane and Women's Hospital; C. Patel, Genetic Health Queensland; C. Simons and J. Crawford, University of Queensland; B. Bennetts, G. Ho, and K. Holman, Childrens Hospital Westmead; and A. Nandini and team, Pathology Queensland, acting as part of the KidGen Collaborative; for clinical and genetic evaluation, mutation identification, and recruitment of fibroblasts from RG_0120.153 (patient with HNF4A mutation). This work was supported by the National Institutes of Health (DK107344-01) and the National Health and Medical Research Council (NHMRC; GNT1098654 and GNT1100970). M.H.L. is a Senior Principal Research Fellow of the NHMRC (GNT1042093). The Murdoch Children's Research Institute is supported by the Victorian Government's Operational Infrastructure Support Program.

Author information

Affiliations

  1. Murdoch Children's Research Institute, The Royal Children's Hospital, Parkville, Victoria, Australia.

    • Sara E Howden
    •  & Melissa H Little
  2. Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria, Australia.

    • Sara E Howden
    •  & Melissa H Little
  3. Morgridge Institute for Research, Madison, Wisconsin, USA.

    • James A Thomson
  4. Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.

    • James A Thomson
  5. Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, Santa Barbara, California, USA.

    • James A Thomson

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Contributions

S.E.H. and J.A.T. conceived and developed the protocol. S.E.H. prepared the manuscript. J.A.T. and M.H.L. provided supervision and assisted in manuscript preparation.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Sara E Howden.

Supplementary information

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  1. 1.

    Supplementary Table 1

    ODNs for sgRNA plasmids used in Anticipated Results section.

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DOI

https://doi.org/10.1038/nprot.2018.007

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