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Simultaneous reprogramming and gene editing of human fibroblasts

Nature Protocols volume 13, pages 875–898 (2018)Cite this article

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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 (∼6–8 weeks), and cost-effective.

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Figure 1: HDRCas9-Gem facilitates generation of indel-free gene-edited iPSCs.
Figure 2
Figure 3: Generation of MAFP:mTagBFP2 reporter iPSC lines following one-step gene editing/reprogramming of healthy fibroblasts.
Figure 4: Simultaneous reprogramming and genetic correction of fibroblasts from a kidney disease patient with an autosomal dominant mutation in HNF4A.
Figure 5: Generation of iPSC lines with a point mutation in H3F3A following one-step gene editing/reprogramming of healthy fibroblasts.

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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.

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Authors and 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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  1. Sara E Howden
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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.

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Correspondence to Sara E Howden.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

ODNs for sgRNA plasmids used in Anticipated Results section. (PDF 203 kb)

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Howden, S., Thomson, J. & Little, M. Simultaneous reprogramming and gene editing of human fibroblasts. Nat Protoc 13, 875–898 (2018). https://doi.org/10.1038/nprot.2018.007

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