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Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, 38198Article in journal (Refereed) Published
Abstract [en]

Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puro Delta tk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.

Place, publisher, year, edition, pages
Nature Publishing Group, 2016. Vol. 6, 38198
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-130086DOI: 10.1038/srep38198ISI: 000389185500001OAI: oai:DiVA.org:umu-130086DiVA: diva2:1064994
Available from: 2017-01-13 Created: 2017-01-11 Last updated: 2017-01-13Bibliographically approved

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Fonfara, InesCharpentier, Emmanuelle
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Molecular Infection Medicine Sweden (MIMS)Umeå Centre for Microbial Research (UCMR)Department of Molecular Biology (Faculty of Medicine)
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Scientific Reports
Microbiology in the medical area

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CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf