Marker-free coselection for CRISPR-driven genome editing in human cells

Daniel Agudelo; Alexis Duringer; Lusiné Bozoyan; Caroline C. Huard; Sophie Carter; Jeremy Loehr; Dafni Synodinou; Mathieu Drouin; Jayme Salsman; Graham Dellaire; Joseé Laganière; Yannick Doyon

doi:10.1038/nmeth.4265

Marker-free coselection for CRISPR-driven genome editing in human cells

Daniel Agudelo, Alexis Duringer, Lusiné Bozoyan, Caroline C. Huard, Sophie Carter, Jeremy Loehr, Dafni Synodinou, Mathieu Drouin, Jayme Salsman, Graham Dellaire, Joseé Laganière, Yannick Doyon

Medicine

Résultat de recherche: Article › examen par les pairs

120 Citations (Scopus)

Résumé

Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems. Selection for dominant alleles of the ubiquitous sodium/potassium pump (Na⁺/K⁺ ATPase) that rendered cells resistant to ouabain was used to enrich for custom genetic modifications at another unlinked locus of interest, thereby effectively increasing the recovery of engineered cells. The process is readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells. The use of universal CRISPR reagents and a commercially available small-molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.

Langue d'origine	English
Pages (de-à)	615-620
Nombre de pages	6
Journal	Nature Methods
Volume	14
Numéro de publication	6
DOI	https://doi.org/10.1038/nmeth.4265
Statut de publication	Published - mai 30 2017

Note bibliographique

Funding Information:
This study was supported by grants from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2014-059680) to Y.D. and the Canadian Institutes of Health Research (CIHR; MOP-84260) to G.D. Salary support was provided by the Fonds de la Recherche du Québec-Santé (FRQS) to D.A. and Y.D. and by a Bridge Grant from the Beatrice Hunter Cancer Research Institute (BHCRI) to J.S. Partial salary support to L.B. was obtained through the Mitacs Accelerate program.

Publisher Copyright:
© 2017 Nature America, Inc. All rights reserved.

ASJC Scopus Subject Areas

Biotechnology
Biochemistry
Molecular Biology
Cell Biology

PubMed: MeSH publication types

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10.1038/nmeth.4265

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abstract = "Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems. Selection for dominant alleles of the ubiquitous sodium/potassium pump (Na+/K+ ATPase) that rendered cells resistant to ouabain was used to enrich for custom genetic modifications at another unlinked locus of interest, thereby effectively increasing the recovery of engineered cells. The process is readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells. The use of universal CRISPR reagents and a commercially available small-molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.",

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Marker-free coselection for CRISPR-driven genome editing in human cells

Résumé

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ASJC Scopus Subject Areas

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