Journal article
Simultaneous reprogramming and gene editing of human fibroblasts
SE Howden, JA Thomson, MH Little
Nature Protocols | NATURE PUBLISHING GROUP | Published : 2018
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 iPSClines from human fibroblasts in the absence of drug selection or FACSenrichment. Using enhanced episomal-based reprogramming and CRISPR/Cas9 systems, gene-edited and passage-matched unmodified iPSClines are obtained following a single electroporation of human fibroblasts. To minimize unwanted mu..
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Awarded by National Institutes of Health
Funding 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.