Simultaneous reprogramming and gene editing of human fibroblasts View Full Text


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Article Info

DATE

2018-04-05

AUTHORS

Sara E Howden, James A Thomson, Melissa H Little

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. More... »

PAGES

875

References to SciGraph publications

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/nprot.2018.007

    DOI

    http://dx.doi.org/10.1038/nprot.2018.007

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1103156237

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/29622803


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    Download the RDF metadata as:  json-ld nt turtle xml License info

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    JSON-LD is a popular format for linked data which is fully compatible with JSON.

    curl -H 'Accept: application/ld+json' 'https://scigraph.springernature.com/pub.10.1038/nprot.2018.007'

    N-Triples is a line-based linked data format ideal for batch operations.

    curl -H 'Accept: application/n-triples' 'https://scigraph.springernature.com/pub.10.1038/nprot.2018.007'

    Turtle is a human-readable linked data format.

    curl -H 'Accept: text/turtle' 'https://scigraph.springernature.com/pub.10.1038/nprot.2018.007'

    RDF/XML is a standard XML format for linked data.

    curl -H 'Accept: application/rdf+xml' 'https://scigraph.springernature.com/pub.10.1038/nprot.2018.007'


     

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