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Nat Methods. 2017 Jun;14(6):615-620. doi: 10.1038/nmeth.4265. Epub 2017 Apr 17.

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

Author information

1
Centre Hospitalier Universitaire de Québec Research Center-Université Laval, Quebec City, Quebec, Canada.
2
Research and Development, Héma-Québec, Quebec City, Quebec, Canada.
3
Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada.

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.

PMID:
28417998
DOI:
10.1038/nmeth.4265
[Indexed for MEDLINE]

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