(A) Schematic representation of epitope knock-in strategy. A crRNA was designed against the 3’UTR of each target gene. Target site with double-stranded break is shown with Cas9 RNP (grey), PAM in yellow box, and single-stranded donor DNA that harbours PAM-blocking mutations and V5 tag coding sequence flanked by 70-mer homology arms on both sides. (B) Cas9 RNP complexes were assembled in vitro by incubation of recombinant Cas9 protein with either IVT sgRNA or synthetic two-part cr:tracrRNA and electroporated into NS cells. V5 ICC was used to quantify knock-in. (C) Representative ICC images for the detection of Olig2-V5 fusion protein in the bulk populations of transfected cells. (D) HDR-mediated insertion of V5 tag was determined by scoring V5-positive cells (%) in the bulk populations of transfected cells. Results from three independent experiments are shown for Sox2 and Olig2 V5 tagging using mouse neural stem (NS) and glioma-initiating neural stem (GNS) cells. Error bars indicate standard deviation values based on a minimum of two experiments, p-values were derived using unpaired t test. (E) ICC for Sox2 gene epitope tagging at the C-terminus with V5, HA, 3XFLAG, or Myc epitope. Numbers represent percentage of tagged cells in the bulk population for each tagging experiment. (F) Representative bulk population V5 ICC images for Sox2, Sox3, Sox8, and Sox9 V5 knock-in are shown. Average knock-in efficiency from two independent experiments is shown at the bottom (numbers in white).