RT Journal Article
SR Electronic
T1 A marker-free co-selection strategy for high efficiency human genome engineering
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 116251
DO 10.1101/116251
A1 Daniel Agudelo
A1 Lusiné Bozoyan
A1 Alexis Duringer
A1 Caroline C. Huard
A1 Sophie Carter
A1 Jeremy Loehr
A1 Dafni Synodinou
A1 Mathieu Drouin
A1 Jayme Salsman
A1 Graham Dellaire
A1 Josée Laganière
A1 Yannick Doyon
YR 2017
UL http://biorxiv.org/content/early/2017/03/15/116251.abstract
AB 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.