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

作者：Agudelo Daniel; Duringer Alexis; Bozoyan Lusine; Huard Caroline C; Carter Sophie; Loehr Jeremy; Synodinou Dafni; Drouin Mathieu; Salsman Jayme; Dellaire Graham; Laganiere Josee; Doyon Yannick

来源：Nature Methods, 2017, 14(6): 615-+.

DOI：10.1038/NMETH.4265

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 (N H EJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR Cas9 and Cpfl 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.

• 出版日期2017-6