Fig. 1.

Mouse and human NSCs are amenable to CRISPR/Cas9-mediated gene targeting. (A) Schematic of the experimental strategy for generating constitutive Luciferase (Luc)-GFP expressing mouse and human NSCs by gene targeting at the safe harbour loci Rosa26 or AAVS1. Targeting vectors contained the CAG-Luc-2A-GFP tethered by an IRES to a blasticidin resistance cassette (BSD); the expression cassettes were flanked by ∼1 kb long homology arms. (B) Schematic depiction of the targeting strategy for the Rosa26 (left) and AAVS1 loci (right). Exons are shown as dark grey blocks. Light grey rectangles indicate the location of the homology arms flanking the expression cassette (L-HA, left homology arm; R-HA, right homology arm). CRISPR sgRNA target sites are indicated with yellow triangles. Horizontal arrows indicate genotyping PCR primers used to confirm on-target integration of the expression cassette. (C) Using FACS, targeted cells were enriched on the basis of GFP expression after blastidicin selection. Wild-type non-transfected mouse and human NSCs were used as a control to set gates for cell sorting. SSC, side scatter. (D) PCR-based genotyping using primer sets 1 and 2 (depicted in A) confirmed correct targeted integration of the CAG-Luc-2A-GFP cassette into the Rosa26 and AAVS1 loci. Non-transfected parental cells were used as negative control for the genotyping. (E) Representative live phase contrast and wide-field fluorescence microscopy images of sorted GFP-positive mouse and human NSCs. Scale bar: 100 μm. (F) Luciferase levels were determined using a microplate reader and confirmed functionality of the targeted cassette in both mouse and human cells. (G) Schematic of the Gateway cloning-based strategy for repurposing targeting vectors with different cassettes of interest. In the example shown, the Luc-2A-GFP in the Rosa26 targeting vector is replaced by a Cas9-GFP expression cassette via LR Gateway cloning. (H) Mouse NSCs were transfected with the Rosa26 Cas9-GFP targeting vector and enriched by FACS on the basis of GFP expression. (I) PCR-based genotyping (top) confirms correct insertion of Cas9-GFP expression cassette at the Rosa26 locus; western immunoblotting (bottom) confirms high levels of Cas9 protein expression in a clonal NSC line derived from the GFP-sorted cells.