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  • Eggenschwiler, Reto (author)

Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase

  • Article/chapterEnglish2016

Publisher, publication year, extent ...

  • 2016-12-02
  • Nature Publishing Group,2016
  • electronicrdacarrier

Numbers

  • LIBRIS-ID:oai:DiVA.org:umu-130086
  • https://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-130086URI
  • https://doi.org/10.1038/srep38198DOI
  • http://kipublications.ki.se/Default.aspx?queryparsed=id:134733211URI

Supplementary language notes

  • Language:English
  • Summary in:English

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  • Subject category:ref swepub-contenttype
  • Subject category:art swepub-publicationtype

Notes

  • 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.

Subject headings and genre

Added entries (persons, corporate bodies, meetings, titles ...)

  • Moslem, Mohsen (author)
  • Fráguas, Mariane Serra (author)
  • Galla, Melanie (author)
  • Papp, Oliver (author)
  • Naujock, Maximilian (author)
  • Fonfara, InesUmeå universitet,Molekylär Infektionsmedicin, Sverige (MIMS),Umeå Centre for Microbial Research (UCMR),Institutionen för molekylärbiologi (Medicinska fakulteten),Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin, Germany(Swepub:umu)info0028 (author)
  • Gensch, Ingrid (author)
  • Wähner, Annabell (author)
  • Beh-Pajooh, Abbas (author)
  • Mussolino, Claudio (author)
  • Tauscher, Marcel (author)
  • Steinemann, Doris (author)
  • Wegner, Florian (author)
  • Petri, Susanne (author)
  • Schambach, Axel (author)
  • Charpentier, EmmanuelleUmeå universitet,Molekylär Infektionsmedicin, Sverige (MIMS),Umeå Centre for Microbial Research (UCMR),Institutionen för molekylärbiologi (Medicinska fakulteten)(Swepub:umu)emch0006 (author)
  • Cathomen, Toni (author)
  • Cantz, Tobias (author)
  • Umeå universitetMolekylär Infektionsmedicin, Sverige (MIMS) (creator_code:org_t)

Related titles

  • In:Scientific Reports: Nature Publishing Group62045-2322

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