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.

Targeted genome editing using engineered nucleases facilitates the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Broadly applicable and versatile methods for increasing the levels of gene editing in cell populations remain highly desirable due to the variable efficiency between distinct genomic loci and cell types. Harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems, we designed a simple and robust co-selection strategy for enriching cells harboring either nuclease-driven non-homologous end joining (NHEJ) or homology-directed repair (HDR) events. Selection for dominant alleles of the endogenous sodium-potassium pump (Na+,K+-ATPase) that render cells resistant to ouabain is used to enrich for custom modifications at another unlinked locus of interest, effectively increasing the recovery of engineered cells. The process was readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells (HSPCs). 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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Genome editing using designer nucleases has revolutionized molecular biology by allowing DNA to be inserted, deleted or replaced in the genome of several model organisms. This protocol describes a marker-free co-selection strategy for high efficiency homology-driven and NHEJ-based gene editing in human cells by generating dominant cellular resistance to ouabain, a highly potent plant-derived inhibitor of the Na+,K+ ATPase (1, 2, 3). This scarless coconversion strategy is highly efficient and can yield a stable population of modified cells in 14 days. Techniques relative to sgRNA cloning, cell nucleofection, selection using ouabain and validation of gene disruption/insertion are described.