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Improved bi-allelic...
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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 ...
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2016-12-02
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Nature Publishing Group,2016
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electronicrdacarrier
Numbers
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LIBRIS-ID:oai:DiVA.org:umu-130086
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https://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-130086URI
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https://doi.org/10.1038/srep38198DOI
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http://kipublications.ki.se/Default.aspx?queryparsed=id:134733211URI
Supplementary language notes
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Language:English
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Summary in:English
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Subject category:ref swepub-contenttype
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Subject category:art swepub-publicationtype
Notes
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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.
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Moslem, Mohsen
(author)
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Fráguas, Mariane Serra
(author)
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Galla, Melanie
(author)
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Papp, Oliver
(author)
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Naujock, Maximilian
(author)
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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)
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Gensch, Ingrid
(author)
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Wähner, Annabell
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Beh-Pajooh, Abbas
(author)
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Mussolino, Claudio
(author)
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Tauscher, Marcel
(author)
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Steinemann, Doris
(author)
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Wegner, Florian
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Petri, Susanne
(author)
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Schambach, Axel
(author)
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Charpentier, EmmanuelleUmeå universitet,Molekylär Infektionsmedicin, Sverige (MIMS),Umeå Centre for Microbial Research (UCMR),Institutionen för molekylärbiologi (Medicinska fakulteten)(Swepub:umu)emch0006
(author)
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Cathomen, Toni
(author)
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Cantz, Tobias
(author)
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Umeå universitetMolekylär Infektionsmedicin, Sverige (MIMS)
(creator_code:org_t)
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In:Scientific Reports: Nature Publishing Group62045-2322
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Eggenschwiler, R ...
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Moslem, Mohsen
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Fráguas, Mariane ...
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Galla, Melanie
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Papp, Oliver
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Naujock, Maximil ...
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Fonfara, Ines
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Gensch, Ingrid
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Wähner, Annabell
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Beh-Pajooh, Abba ...
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Mussolino, Claud ...
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Tauscher, Marcel
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Steinemann, Dori ...
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Wegner, Florian
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Petri, Susanne
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Schambach, Axel
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Charpentier, Emm ...
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Cathomen, Toni
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Cantz, Tobias
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Umeå University
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Karolinska Institutet