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

Authors:
Agudelo D, Duringer A, Bozoyan L, Huard CC, Carter S, Loehr J, Synodinou D, Drouin M, Salsman J, Dellaire G, Laganière J, Doyon Y.
In:
Source: Nat Methods
Publication Date: (2017)
Issue: 14(6): 615-620
Research Area:
Gene Expression
Cells used in publication:
K-562
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
293T
Species: human
Tissue Origin: kidney
U-2 OS
Species: human
Tissue Origin: bone
Platform:
4D-Nucleofector™ X-Unit
Experiment

CD34+ HSPCs were thawed and cultured in StemSpan ACF (StemCell Technologies) supplemented with 100 ng/ml SCF (Feldan), 100 ng/ml FLT3-l (Peprotech), 50 ng/ml TPO (StemCell Technologies), 10 µg/ml LDL (StemCell Technologies), and 35 nM UM171 (StemCell Technologies) for 16–24 h after thawing. Cells were then nucleofected with Cas9 RNP and ssODNs with an Amaxa 4D Nucleofector X unit (Lonza) and the E0-100 program, according to the manufacturer’s recommendations. K562, HEK293LTV, RPE-1:Cells (2 × 105 per transfection) were transfected with an Amaxa 4D-Nucleofector (Lonza), per the manufacturer’s recommendations.

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.