Fig. 6.

a CRISPR/Cas9 guide design: the single guide RNA (sgRNA; red square) was chosen to target a genomic region which is conserved between Ago1 and Ago2 but avoiding off-targets. b Western blot of Ago* proteins in control (Ctrl) or Ago1–2 CRISPR ES cells 4 days after transduction. c Western blot of Dnmt3b, SmarcA4, Kdm2b, Nanog, and GAPDH protein levels in ES cells cultured in 2i medium 4 days after transduction with Ago1–2 CRISPR lentiviral vector compared with control cells transduced with non-targeting CRISPR vector (Ctrl CRISPR). d mRNA levels of Dnmt3b, SmarcA4, and Kdm2b of cells as in c. e, f mRNA levels of pluripotency (e) or early neural commitment (f) in cells as in c and d 8 days after transduction. g, h GFP immunodetection in a 46C Sox1::GFP mESC line cultured in 2i medium 8 days after transduction with Ctrl CRISPR (g) or Ago1–2 CRISPR lentiviral vector (h). Scale bars, 50 microns. i, j Cell count (i) and cytofluorimetric analysis (j) of GFP-positive cells as in g and h. *p = 0.05, **p = 0.01, ***p = 0.001 (e–f, REST randomization test; i, Student’s t-test). Error bars show standard error